Nogo receptor binding protein

ABSTRACT

The invention provides Sp35 polypeptides and fusion proteins thereof, Sp35 antibodies and antigen-binding fragments thereof and nucleic acids encoding the same. The invention also provides compositions comprising, and methods for making and using, such Sp35 antibodies, antigen-binding fragments thereof, Sp35 polypeptides and fusion proteins thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage of International ApplicationNo. PCT/US2004/008323, filed Mar. 17, 2004 which claims the benefit ofU.S. Provisional Application No. 60/455,756, filed Mar. 19, 2003, U.S.Provisional Application No. 60/480,241, filed Jun. 20, 2003, and U.S.Provisional Application No. 60/492,057, filed Aug. 1, 2003, all of whichare hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to neurology, neurobiology and molecular biology.More particularly, this invention relates to molecules and methods fortreatment of neurological diseases, disorders and injuries such asspinal cord injury.

BACKGROUND OF THE INVENTION

Axons and dendrites extend from neurons. The distal tip of an extendingaxon or neurite includes a specialized region, known as the growth cone.Growth cones sense the local environment and guide axonal growth towarda neuron's target cell. Growth cones respond to environmental cues, forexample, surface adhesiveness, growth factors, neurotransmitters andelectric fields. The growth cones generally advance at a rate of one totwo millimeters per day. The growth cone explores the area ahead of itand on either side, by means of elongations classified as lamellipodiaand filopodia. When an elongation contacts an unfavorable surface, itwithdraws. When an elongation contacts a favorable growth surface, itcontinues to extend and guides the growth cone in that direction. Whenthe growth cone reaches an appropriate target cell a synaptic connectionis created.

Nerve cell function is influenced by contact between neurons and othercells in their immediate environment (Rutishauser, et al., 1988,Physiol. Rev. 68:819). These cells include specialized glial cells,oligodendrocytes in the central nervous system (CNS), and Schwann cellsin the peripheral nervous system (PNS), which sheathe the neuronal axonwith myelin (Lemke, 1992, in An Introduction to Molecular Neurobiology,Z. Hall, Ed., p. 281, Sinauer).

CNS neurons have the inherent potential to regenerate after injury, butthey are inhibited from doing so by inhibitory proteins present inmyelin (Brittis et al., 2001, Neuron 30:11-14; Jones et al, 2002, J.Neurosci. 22:2792-2803; Grimpe et al, 2002, J. Neurosci.:22:3144-3160).

Several myelin inhibitory proteins found on oligodendrocytes have beencharacterized. Known examples of myelin inhibitory proteins includeNogoA (Chen et al., Nature, 2000, 403, 434-439; Grandpre et al., Nature2000, 403, 439-444), myelin associated glycoprotein (MAG) (McKerracheret al., 1994, Neuron 13:805-811; Mukhopadhyay et al., 1994, Neuron13:757-767) and oligodendrocyte glycoprotein (OM-gp), Mikol et al.,1988, J. Cell. Biol. 106:1273-1279). Each of these proteins has beenseparately shown to be a ligand for the neuronal NgR1 (Wang et al.,Nature 2002, 417, 941-944; Grandpre et al., Nature 2000, 403, 439-444;Chen et al., Nature, 2000, 403, 434-439; Domeniconi et al., Neuron 2002,published online Jun. 28, 2002).

Nogo receptor-1 (NgR1) is a GPI-anchored membrane protein that contains8 leucine rich repeats (Fournier et al., 2001, Nature 409:341-346). Uponinteraction with inhibitory proteins (e.g., NogoA, MAG and OM-gp), theNgR1 complex transduces signals that lead to growth cone collapse andinhibition of neurite outgrowth.

There is an unmet need for molecules and methods for inhibitingNgR1-mediated growth cone collapse and the resulting inhibition ofneurite outgrowth.

SUMMARY OF THE INVENTION

We have made various discoveries regarding a polypeptide designated“Sp35” (our designation). Alternate designations for Sp35 include“LINGO” and “LINGO-1.” Our discoveries include the following. Sp35 bindsto NgR1. Sp35 binds to itself in a homotypic interaction. An Sp35-Fcfusion protein induces or promotes fasciculation in granular neurons. AnSp35-Fc fusion protein promotes neuronal survival in both therubro-spinal tract hemisection injury model and the optic nervetransection model. Sp35 retrovirus-infected cortical primary cells, whendelivered into spinal cord-injured rats, result in enhanced neuronsurvival, increased β III tubulin staining of axons, and increasedmyelin content.

Based in part on these discoveries, the invention features an isolatednucleic acid containing a nucleotide sequence encoding a polypeptidewherein: (a) the polypeptide includes (i) an Sp35 LRR domain, (ii) anSp35 basic region C-terminal to the LRR domain, and (iii) an Sp35immunoglobulin (Ig) domain C-terminal to the basic region; and (b) thepolypeptide lacks a transmembrane domain. The Sp35 LRR domain cancontain a carboxy-terminal LRR (LRRCT), an amino-terminal LRR (LRRNT),or both. In some embodiments of the invention, the encoded Sp35polypeptide lacks the cytoplasmic domain. In some embodiments, theencoded Sp35 polypeptide includes amino acid residues 34-532 of SEQ IDNO: 2 and lacks amino acid residues 533-614.

The invention also includes a nucleic acid encoding a polypeptidewherein the polypeptide includes an Sp35 Ig domain and lacks an Sp35 LRRdomain, an Sp35 basic region, a transmembrane domain, and a cytoplasmicdomain.

The invention also includes a nucleic acid encoding a polypeptidewherein the polypeptide includes an Sp35 LRR domain and lacks an Sp35 Igdomain, an Sp35 basic region, a transmembrane domain, and a cytoplasmicdomain.

The invention also includes a nucleic acid encoding a polypeptidelacking a functional cytoplasmic domain but including all the other Sp35domains. For example, the encoded polypeptide could include amino acids1-576 of SEQ ID NO: 2 (prior to processing of the signal sequence).

In some embodiments of the invention, the encoded polypeptide is afusion polypeptide containing a non-Sp35 moiety. The non-Sp35 moiety canbe, for example, an Ig moiety, a serum albumin moiety, a targetingmoiety, a reporter moiety, or a purification-facilitating moiety. Apreferred non-Sp35 moiety is an Ig moiety, e.g., an Fc moiety.

The nucleotide sequence can be operatively linked to an expressioncontrol sequence, for example, in an expression vector. The inventionalso includes a host cell transformed with a vector that expresses anSp35 polypeptide of the invention.

The invention also includes an Sp35 polypeptide encoded by any of theabove-described nucleic acids.

The invention also includes an Sp35 polypeptide conjugated to a polymer,e.g., a polyalkylene glycol, a sugar polymer, and a polypeptide. Apreferred polymer is a polyalkylene glycol, e.g., polyethylene glycol(PEG). The polypeptide can be conjugated to 1, 2, 3 or 4 polymers.Preferably, the total molecular weight of the conjugated polymers isfrom 20,000 Da to 40,000 Da per Sp35 polypeptide.

The invention also includes a method of inhibiting signal transductionby NgR1. The method includes contacting the NgR1 with an effectiveamount of an Sp35 polypeptide. Preferred polypeptides for use in themethod include the following:

-   -   (a) an Sp35 polypeptide, wherein: (a) the polypeptide        includes (i) an Sp35 LRR domain, (ii) an Sp35 basic region        C-terminal to the LRR domain, and (iii) an Sp35 immunoglobulin        (Ig) domain C-terminal to the basic region; and (b) the        polypeptide lacks a transmembrane domain; and    -   (b) an Sp35 polypeptide that includes an Sp35 Ig domain and        lacks an Sp35 LRR domain, an Sp35 basic region, a transmembrane        domain, and a cytoplasmic domain.

The invention also includes a method of decreasing inhibition of axonalgrowth of a central nervous system (CNS) neuron. The method includescontacting the neuron with an effective amount of a polypeptide such asan Sp35 polypeptide, an anti-Sp35 antibody, or an antigen-bindingfragment of an anti-Sp35 antibody.

The invention also includes a method of inhibiting growth cone collapseof a CNS neuron. The method includes contacting the neuron with aneffective amount of a polypeptide such as an Sp35 polypeptide, ananti-Sp35 antibody, or an antigen-binding fragment of an anti-Sp35antibody.

The invention also includes a method of treating a CNS disease, disorderor injury in a mammal. The method includes administering to the mammal atherapeutically effective amount of a polypeptide such as an Sp35polypeptide, an anti-Sp35 antibody, or an antigen-binding fragment of ananti-Sp35 antibody. In some embodiments of the invention, the CNSdisease, disorder or injury is a spinal cord injury. The Sp35polypeptide can be administered locally. In some embodiments of themethod, the Sp 35 polypeptide is administered initially within 48 hoursof a spinal cord injury. For local administration, the therapeuticallyeffective amount of the polypeptide preferably is from 10 μg/kg to 10mg/kg. For systemic administration, the therapeutically effective amountof the polypeptide preferably is from 1 mg/kg to 20 mg/kg.

The invention also includes an ex vivo gene therapy method of treating aCNS disease, disorder or injury in a mammal. The method includes (a)providing a cultured host cell expressing a recombinant Sp35polypeptide; and (b) introducing the host cell into the mammal at thesite of the CNS disease, disorder or injury, e.g., spinal cord injury.The cultured host cell can be derived from the mammal to be treated. Inthis ex vivo gene therapy method, the recombinant Sp35 polypeptide canbe a full-length Sp35 polypeptide.

The invention also includes a method of promoting myelination at thesite of the CNS disease, disorder or injury. The method includescontacting the site of the CNS disease, disorder or injury with aneffective amount of an Sp35 polypeptide, e.g., a polypeptide containingan Sp35 LRR domain and lacking an Sp35 Ig domain, an Sp35 basic region,a transmembrane domain, and a cytoplasmic domain.

The invention also includes an in vivo gene therapy method of treating aCNS disease, disorder or injury by in vivo gene therapy. The methodincludes the steps of administering to a mammal, at or near the site ofthe disease, disorder or injury, a viral vector containing a nucleotidesequence that encodes an Sp35 polypeptide so that the Sp35 polypeptideis expressed from the nucleotide sequence in the mammal in an amountsufficient to reduce inhibition of axonal extension by neurons at ornear the site of the injury. The viral vector can be, e.g., anadenoviral vector, a lentiviral vector, a baculoviral vector, an EpsteinBarr viral vector, a papovaviral vector, a vaccinia viral vector, and aherpes simplex viral vector. The disease, disorder or injury can be,e.g., spinal cord injury or optic nerve injury. The viral vector can beadministered by a route such as topical administration, intraocularadministration, parenteral administration, intrathecal administration,subdural administration and subcutaneous administration.

The invention also includes a method of promoting survival of a neuronat risk of dying. The method includes contacting the neuron with aneffective amount of an Sp35 polypeptide. The Sp35 polypeptide can be asoluble form of Sp35, e.g., an Sp35-Fc fusion protein. The neuron can bein vitro or in vivo, e.g., in a mammal with a neurodegenerative diseasedisorder or injury, e.g., multiple sclerosis, ALS, Huntington's disease,Alzheimer's disease, Parkinson's disease, diabetic neuropathy, stroke,traumatic brain injuries and spinal cord injury. In some embodiments ofthe invention, the Sp35 polypeptide is administered indirectly by: (a)providing a cultured host cell expressing a recombinant Sp35polypeptide; and (b) introducing the host cell into the mammal at thesite of the neuron. In some embodiments of the invention, thepolypeptide is administered indirectly through in vivo gene therapy. Insuch an embodiment, the method includes administering, at or near thesite of the neuron, a viral vector comprising a nucleotide sequence thatencodes an Sp35 polypeptide so that the Sp35 polypeptide is expressedfrom the nucleotide sequence in the mammal in an amount sufficient topromote survival of the neuron.

As used herein, “full length human Sp35 polypeptide” means thepolypeptide whose amino acid sequence is amino acids 34-614 of SEQ IDNO: 2.

As used herein, “heterologous moiety” means an amino acid sequence notpresent in a full-length Sp35 polypeptide.

As used herein, “nogo receptor-1” means the polypeptide whose sequenceis publicly available under Genbank accession no. AAG53612.

As used herein, “Sp35 antagonist polypeptide” means an Sp35 polypeptidethat blocks, inhibits, or interferes with the biological activity ofnaturally-occurring Sp35.

As used herein, “Sp35 basic region” means the following amino acidmotif:

RRARIRDRK (SEQ ID NO: 4) KKVKVKEKR (SEQ ID NO: 5) RRLRLRDRK (SEQ ID NO:6) RRGRGRDRK (SEQ ID NO: 7) RRIRARDRK (SEQ ID NO: 8)The top row of amino acids (in bold; SEQ ID NO: 4) is the preferred Sp35basic region sequence, with variants showing optional substitutionsshown below (SEQ ID NOS:5,6,7 and 8).

As used herein, “Sp35 fusion protein” means a fusion protein thatincludes an Sp35 moiety fused to a heterologous moiety.

As used herein, “Sp35 Ig domain” means amino acids 433-493 of SEQ ID NO:2, provided that the sequence can contain up to five individual aminoacid insertions, deletions, or conservative amino acid substitutions.The following substitutions (numbering based on SEQ ID NO: 2) areexpressly included: V to M at position 6; S to G at position 294; V to Aat position 348; and R to H at position 419.

As used herein, “Sp35 LRR domain” means a domain that includes 10 to 14of the leucine rich repeat sequences, including the LRRNT and LRRCT,listed in Table 1, provided that up to five amino acid insertions,deletions, or conservative amino acid substitutions can appear withinthe aggregate 10-14 leucine rich repeats.

As used herein, “Sp35 moiety” means a biologically active fragment of afull-length Sp35 polypeptide.

As used herein, “Sp35 polypeptide” means an Sp35 moiety or a fusionprotein that includes an Sp35 moiety.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. In case of conflict, thepresent specification, including definitions, will control. Allpublications, patents and other references mentioned herein areincorporated by reference.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the invention, thepreferred methods and materials are described below. The materials,methods and examples are illustrative only, and are not intended to belimiting. Other features and advantages of the invention will beapparent from the detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleotide sequence of a full length human Sp35 cDNA (SEQID NO: 1)

FIG. 2 is the amino acid sequence of a full-length human Sp35polypeptide (SEQ ID NO: 2).

FIG. 3 is a schematic illustration of the Sp35 domain structure anddeletion mapping to identify Sp35 sequence(s) that bind to NgR1.

FIG. 4 is a histogram summarizing data on SP35 binding to COS7 cellstransfected with an expression vector encoding rat p75 or a vectorcontrol. After 48 hours, AP-SP35 or AP was incubated with the cells.Bound AP was detected using chromogenic AP detection reagent.

FIG. 5 is a histogram summarizing data on binding of AP-Omgp, andAP-Nogo-66 to COS7 cells transfected with an expression vector encodingNgR1; NgR1 and p75; NgR1, p75, and SP35, or a vector control. After 48hours, AP-Omgp, AP-Nogo-66 or AP was incubated with the cells. Bound APwas detected using chromogenic AP detection reagent.

FIG. 6 is a histogram summarizing data on the relief of inhibitoryactivity of myelin inhibitors on neurite outgrowth in vitro. Neuritelength was measured on postnatal day 7 rat cerebellar granular neuronsexpressing DN-Sp35, full-length Sp35, or controls cultured onimmobolized substrate Omgp, Myelin and Nogo-66. DN-SP35 transfectedcells exhibited diminished response to inhibitory substrates. Neuritelength was quantified from 1000 neurons per treatment group from twoindependent experiments (p<0.01).

FIG. 7 is a histogram summarizing data on reversal of inhibitoryactivity of myelin inhibitors by SP35-Fc. Neurite length of postnatalday 7 rat cerebellar granular neurons (1000 neurons) cultured onimmobilized substrate Omgp, Myelin or Nogo-66 in the presence or absenceof SP35-Fc. SP35-Fc reduced the neurite outgrowth inhibition caused byOmgp, Nogo-66 and MAG. Neurite length was quantified from 1000 neuronsper treatment group from two independent experiments (p<0.01).

FIG. 8 is a graph summarizing data from an experiment showing thatintrathecal-administered Sp35-Fc improves functional recovery afterdorsal hemisection in a rat. The locomoter BBB score measured as afunction of time after dorsal hemisection in control (IgG) orSp35-Fc-treated rats (8 animals per group). Treatment was initiated atthe time of spinal cord injury.

FIG. 9 is a graph showing individual animal BBB scores at week four inthe experiment summarized in FIG. 8.

DETAILED DESCRIPTION OF THE INVENTION

Naturally occurring human Sp35 is a glycosylated CNS-specific proteincontaining 614 amino acids (FIG. 2; SEQ ID NO: 2). The human,full-length wild-type SP35 polypeptide contains an LRR domain consistingof 14 leucine-rich repeats (including N- and C-terminal caps), an Igdomain, a transmembrane region, and a cytoplasmic domain (FIG. 3). Thecytoplasmic domain contains a canonical tyrosine phosphorylation site.In addition, the naturally occurring Sp35 protein contains a signalsequence, a short basic region between the LRRCT and Ig domain, and atransmembrane region between the Ig domain and the cytoplasmic domain(FIG. 3). The human Sp35 gene contains alternative translation startcodons, so that six additional amino acids, i.e., MQVSKR (SEQ ID NO: 9)may or may not be present at the N-terminus of the Sp35 signal sequence.Table 1 lists the Sp35 domains and other regions, according to aminoacid residue number, based on the sequence in FIG. 2 (SEQ ID NO: 2).

TABLE 1 Domain or Region Beginning Residue Ending Residue SignalSequence 1 33 LRRNT 34 64 LRR 66 89 LRR 90 113 LRR 114 137 LRR 138 161LRR 162 185 LRR 186 209 LRR 210 233 LRR 234 257 LRR 258 281 LRR 282 305LRR 306 329 LRR 330 353 LRRCT 363 416 Basic 417 424 Ig 433 493Connecting sequence 494 551 Transmembrane 552 576 Cytoplasmic 577 614

Tissue distribution and developmental expression of Sp35 has beenstudied in humans and rats. Sp35 biology has been studied in anexperimental animal (rat) model. Expression of rat SP35 is localized toCNS neurons and brain oligodendrocytes, as determined by northern blotand immuno-histochemical staining. Rat Sp35 mRNA expression level isregulated developmentally, peaking shortly after birth, i.e., ca.postnatal day one. In a rat spinal cord transection injury model, Sp35is up-regulated at the injury site, as determined by RT-PCR.

The inventors have discovered that full-length, wild-type Sp35 binds toNgR1. Soluble derivatives of Sp35 function as Sp35 antagonistpolypeptides by binding to NgR1 and blocking, inhibiting, or interferingwith its function, thereby relieving the NgR1-mediated inhibition ofaxonal extension that normally takes place in CNS neurons. This isbeneficial in situations where axonal extension or neurite sprouting isneeded in the brain or spinal cord. Spinal cord injury, includingpartial or complete crush or severance, exemplifies a situation in whichaxonal extension is needed, but is normally inhibited through operationof the Nogo pathway. Examples of diseases or disorders in which axonalextension and/or neurite sprouting in the brain would be beneficialinclude stroke, multiple sclerosis, and other neurodegenerative diseasesor disorders.

In methods of the present invention, an Sp35 polypeptide or an Sp35blocking antibody (or antigen-binding antibody fragment) can beadministered directly as a preformed polypeptide, or indirectly througha nucleic acid vector, to antagonize NgR1 function and permit beneficialaxonal outgrowth.

In some embodiments of the invention a soluble Sp35 antagonistpolypeptide is administered in a treatment method that includes: (1)transforming or transfecting an implantable host cell with a nucleicacid, e.g., a vector, that expresses an Sp35 polypeptide; and (2)implanting the transformed host cell into a mammal, at the site of adisease, disorder or injury. For example, the transformed host cell canbe implanted at the site of a spinal cord injury. In some embodiments ofthe invention, the implantable host cell is removed from a mammal,temporarily cultured, transformed or transfected with an isolatednucleic acid encoding a soluble Sp35 polypeptide, and implanted backinto the same mammal from which it was removed. The cell can be, but isnot required to be, removed from the same site at which it is implanted.Such embodiments, sometimes known as ex vivo gene therapy, can provide acontinuous supply of the Sp35 polypeptide, localized at the site of siteof action, for a limited period of time.

The invention provides oligopeptides useful as modulators of the Sp35interaction with NgR1 and Sp35 homotypic interactions. The oligopeptidesinclude the following amino acid motif:

LSPRKH (SEQ ID NO: 10) ITPKRR (SEQ ID NO: 11) ACPHHK (SEQ ID NO: 12)VSPRKH (SEQ ID NO: 13)

The top row of amino acids (in bold; SEQ ID NO: 10) is the preferredsequence, with variants comprising optional substitutions shown below(SEQ ID NOS: 11, 12 and 13).

Various exemplary Sp35 polypeptides, anti-Sp35 antibodies and antibodyfragments, and methods and materials for obtaining these molecules forpracticing the present invention are described below.

Fusion Proteins and Conjugated Polypeptides

Some embodiments of the invention involve the use of an Sp35polypeptide, e.g., an Sp35 antagonist polypeptide, wherein an Sp35moiety is fused to a heterologous polypeptide moiety to form an Sp35fusion protein. Sp35 fusion proteins can be used to accomplish variousobjectives, e.g., increased serum half-life, improved bioavailability,in vivo targeting to a specific organ or tissue type, improvedrecombinant expression efficiency, improved host cell secretion, ease ofpurification, and higher avidity. Depending on the objective(s) to beachieved, the heterologous moiety can be inert or biologically active.Also, it can be chosen to be stably fused to the Sp35 moiety or to becleavable, in vitro or in vivo. Heterologous moieties to accomplishdifferent objectives are known in the art.

As an alternative to expression of a Sp35 fusion protein, a chosenheterologous moiety can be preformed and chemically conjugated to theSp35 moiety. In most cases, a chosen heterologous moiety will functionsimilarly, whether fused or conjugated to the Sp35 moiety. Therefore, inthe following discussion of heterologous amino acid sequences, unlessotherwise noted, it is to be understood that the heterologous sequencecan be joined to the Sp35 moiety in the form of a fusion protein or as achemical conjugate.

Pharmacologically active polypeptides such as Sp35 often exhibit rapidin vivo clearance, necessitating large doses to achieve therapeuticallyeffective concentrations in the body. In addition, polypeptides smallerthan about 60 kDa potentially undergo glomerular filtration, whichsometimes leads to nephrotoxicity. Fusion or conjugation of relativelysmall polypeptides such as Sp35 fragments can be employed to reduce oravoid the risk of such nephrotoxicity. Various heterologous amino acidsequences, i.e., polypeptide moieties or “carriers,” for increasing thein vivo stability, i.e., serum half-life, of therapeutic polypeptidesare known.

Due to its long half-life, wide in vivo distribution, and lack ofenzymatic or immunological function, essentially full-length human serumalbumin (HSA), or an HSA fragment, is a preferred heterologous moiety.Through application of methods and materials such as those taught in Yehet al., 1992, Proc. Natl. Acad. Sci. USA, 89:1904-1908 and Syed et al.,1997, Blood 89:3243-3252, HSA can be used to form an Sp35 fusion proteinor conjugate that displays pharmacological activity by virtue of theSp35 moiety while displaying significantly increased, e.g., 10-fold to100-fold higher, in vivo stability. Preferably, the C-terminus of theHSA is fused to the N-terminus of the Sp35 moiety. Since HSA is anaturally secreted protein, the HSA signal sequence can be exploited toobtain secretion of the Sp35 fusion protein into the cell culturemedium, when the fusion protein is produced in a eukaryotic, e.g.,mammalian, expression system.

Some embodiments of the invention employ an Sp35 polypeptide wherein anSp35 moiety is fused to an Fc region, i.e., the C-terminal portion of anIg heavy chain constant region. Potential advantages of an Sp35-Fcfusion include solubility, in vivo stability, and multivalency, e.g.,dimerization. The Fc region used can be an IgA, IgD, or IgG Fc region(hinge-CH2-CH3). Alternatively, it can be an IgE or IgM Fc region(hinge-CH2-CH3-CH4). An IgG Fc region is preferred, e.g., an IgG1 Fcregion or IgG4 Fc region. Materials and methods for constructing andexpressing DNA encoding Fc fusions are known in the art and can beapplied to obtain Sp35 fusions without undue experimentation. Someembodiments of the invention employ an Sp35 fusion protein such as thosedescribed in Capon et al. U.S. Pat. Nos. 5,428,130 and 5,565,335.

The signal sequence is a polynucleotide that encodes an amino acidsequence that initiates transport of a protein across the membrane ofthe endoplasmic reticulum. Signal sequences useful for constructing animmunofusin include antibody light chain signal sequences, e.g.,antibody 14.18 (Gillies et. al., 1989, J. Inmunol. Meth., 125:191-202),antibody heavy chain signal sequences, e.g., the MOPC141 antibody heavychain signal sequence (Sakano et al., 1980, Nature 286:5774).Alternatively, other signal sequences can be used. See, for example,Watson, 1984, Nucleic Acids Research 12:5145). The signal peptide isusually cleaved in the lumen of the endoplasmic reticulum by signalpeptidases. This results in the secretion of a immunofusin proteincontaining the Fc region and the Sp35 moiety.

In some embodiments the DNA sequence encodes a proteolytic cleavage sitebetween the secretion cassette and the Sp35 moiety. A cleavage siteprovides for the proteolytic cleavage of the encoded fusion protein,thus separating the Fc domain from the target protein. Usefulproteolytic cleavage sites include amino acids sequences recognized byproteolytic enzymes such as trypsin, plasmin, thrombin, factor Xa, orenterokinase K.

The secretion cassette can be incorporated into a replicable expressionvector. Useful vectors include linear nucleic acids, plasmids,phagemids, cosmids and the like. An exemplary expression vector is pdC,in which the transcription of the immunofusin DNA is placed under thecontrol of the enhancer and promoter of the human cytomegalovirus. See,e.g., Lo et al., 1991, Biochim. Biophys. Acta 1088:712; and Lo et al.,1998, Protein Engineering 11:495-500. An appropriate host cell can betransformed or transfected with a DNA that encodes an Sp35 polypeptide,and is used for the expression and secretion of the Sp35 polypeptide.Preferred host cells include immortal hybridoma cells, myeloma cells,293 cells, Chinese hamster ovary (CHO) cells, Hela cells, and COS cells.

Fully intact, wild-type Fc regions display effector functions thatnormally are unnecessary and undesired in an Fc fusion protein accordingto the present invention. Therefore, certain binding sites preferablyare deleted from the Fc region during the construction of the secretioncassette. For example, since coexpression with the light chain isunnecessary, the binding site for the heavy chain binding protein, Bip(Hendershot et al., 1987, Immunol. Today 8:111-114), is deleted from theCH2 domain of the Fc region of IgE, such that this site does notinterfere with the efficient secretion of the immunofusin. Likewise, thecysteine residues present in the Fc regions which are responsible forbinding to the light chain of the immunoglobulin should be deleted orsubstituted with another amino acid, such that these cysteine residuesdo not interfere with the proper folding of the Fc region when it isproduced as an immunofusin. Transmembrane domain sequences, such asthose present in IgM, should be deleted.

The IgG1 Fc region is preferred. Alternatively, the Fc region of theother subclasses of immunoglobulin gamma (gamma-2, gamma-3 and gamma-4)can be used in the secretion cassette. The IgG1 Fc region ofimmunoglobulin gamma-1 is preferably used in the secretion cassetteincludes the hinge region (at least part), the CH2 region, and the CH3region. In some embodiments, the Fc region of immunoglobulin gamma-1 isa CH2-deleted-Fc, which includes part of the hinge region and the CH3region, but not the CH2 region. A CH2-deleted-Fc has been described byGillies et al., 1990, Hum. Antibod. Hybridomas, 1:47. In someembodiments, the Fc regions of IgA, IgD, IgE, or IgM, are used.

Sp35-Fc fusion proteins can be constructed in several differentconfigurations. In one configuration the C-terminus of the Sp35 moietyis fused directly to the N-terminus of the Fc moiety. In a slightlydifferent configuration, a short polypeptide, e.g., 2-10 amino acids, isincorporated into the fusion between the N-terminus of the Sp35 moietyand the C-terminus of the Fc moiety. Such a linker providesconformational flexibility, which may improve biological activity insome circumstances. If a sufficient portion of the hinge region isretained in the Fc moiety, the Sp35-Fc fusion will dimerize, thusforming a divalent molecule. A homogeneous population of monomeric Fcfusions will yield monospecific, bivalent dimers. A mixture of twomonomeric Fc fusions each having a different specificity will yieldbispecific, bivalent dimers.

Any of a number of cross-linkers that contain a corresponding aminoreactive group and thiol reactive group can be used to link Sp35 toserum albumin. Examples of suitable linkers include amine reactivecross-linkers that insert a thiol reactive-maleimide, e.g., SMCC, AMAS,BMPS, MBS, EMCS, SMPB, SMPH, KMUS, and GMBS. Other suitable linkersinsert a thiol reactive-haloacetate group, e.g., SBAP, SIA, SIAB.Linkers that provide a protected or non-protected thiol for reactionwith sulfhydryl groups to product a reducible linkage include SPDP,SMPT, SATA, and SATP. Such reagents are commercially available (e.g.,Pierce Chemicals).

Conjugation does not have to involve the N-terminus of an Sp35polypeptide or the thiol moiety on serum albumin. For example,Sp35-albumin fusions can be obtained using genetic engineeringtechniques, wherein the Sp35 moiety is fused to the serum albumin geneat its N-terminus, C-terminus, or both.

Sp35 polypeptides can be fused to heterologous peptides to facilitatepurification or identification of the Sp35 moiety. For example, ahistidine tag can be fused to an Sp35 polypeptide to facilitatepurification using commercially available chromatography media.

In some embodiments of the invention, an Sp35 fusion construct is usedto enhance the production of an Sp35 moiety in bacteria. In suchconstructs a bacterial protein normally expressed and/or secreted at ahigh level is employed as the N-terminal fusion partner of an Sp35polypeptide. See, e.g., Smith et al., 1988 Gene 67:31; Hopp et al.,1988, Biotechnology 6:1204; La Vallie et al., 1993, Biotechnology11:187.

In some embodiments of the invention, a fusion construct includes anSp35 moiety and a second human NgR1-binding moiety, e.g., anoligodendrocyte-myelin glycoprotein (OMgp) moiety, a myelin associatedglycoprotein (MAG) moiety, or Nogo66 moiety. Advantages of suchconstructs include increased NgR1 binding affinity.

The full-length OMgp amino acid sequence is known in the art (Genbankaccession no. P23515). Specific examples of Sp35-OMgp fusions includethe following:

Sp35 (aa 34-532)+IgG1 Fc+OMgp (amino acid residues 25-400); and

Sp35 (aa 34-532)+HSA+OMgp (amino acid residues 25-400).

The full-length MAG amino acid sequence is known in the art (Genbankaccession no. A61084). Specific examples of Sp35-MAG fusions include thefollowing:

Sp35 (aa 34-532)+IgG1 Fc+MAG (amino acid residues 12-500); and

Sp35 (aa 34-532)+HSA+MAG (amino acid residues 12-500).

The full-length Nogo amino acid sequence is known in the art (NogoAGenbank accession no. AY102279). Specific examples of Sp35-Nogo fusionsinclude the following:

Sp35 (aa 34-532)+IgG1 Fc+Nogo66 (NogoA amino acid residues 1056-1122);

Sp35 (aa 34-532)+HSA+Nogo66 (NogoA amino acid residues 1056-1122);

Sp35 (aa 34-532)+IgG1 Fc+amino Nogo (NogoA amino acid residues 1-949);and

Sp35 (aa 34-532)+HSA+amino Nogo (NogoA amino acid residues 1-949).

By fusing an Sp35 moiety at the amino and carboxy termini of a suitablefusion partner, bivalent or tetravalent forms of an Sp35 polypeptide canbe obtained. For example, an Sp35 moiety can be fused to the amino andcarboxy termini of an Ig moiety to produce a bivalent monomericpolypeptide containing two Sp35 moieties. Upon dimerization of two ofthese monomers, by virtue of the Ig moiety, a tetravalent form of anSp35 protein is obtained. Such multivalent forms can be used to achieveincreased binding affinity for the target. Multivalent forms of Sp35also can be obtained by placing Sp35 moieties in tandem to formconcatamers, which can be employed alone or fused to a fusion partnersuch as Ig or HSA.

Conjugated Polymers (other than polypeptides)

Some embodiments of the invention involve an Sp35 polypeptide whereinone or more polymers are conjugated (covalently linked) to the Sp35polypeptide. Examples of polymers suitable for such conjugation includepolypeptides (discussed above), sugar polymers and polyalkylene glycolchains. Typically, but not necessarily, a polymer is conjugated to theSp35 polypeptide for the purpose of improving one or more of thefollowing: solubility, stability, or bioavailability.

A preferred class of polymer for conjugation to an Sp35 polypeptide is apolyalkylene glycol. Polyethylene glycol (PEG) is particularlypreferred. PEG moieties, e.g., 1, 2, 3, 4 or 5 PEG polymers, can beconjugated to each Sp35 polypeptide to increase serum half life, ascompared to the Sp35 polypeptide alone. PEG moieties are non-antigenicand essentially biologically inert. PEG moieties used in the practice ofthe invention may be branched or unbranched.

The number of PEG moieties attached to the Sp35 polypeptide and themolecular weight of the individual PEG chains can vary. In general, thehigher the molecular weight of the polymer, the fewer polymer chainsattached to the polypeptide. Preferably, the total polymer mass attachedto the Sp35 polypeptide is from 20 kDa to 40 kDa. Thus, if one polymerchain is attached, the preferred molecular weight of the chain is 20-40kDa. If two chains are attached, the preferred molecular weight of eachchain is 10-20 kDa. If three chains are attached, the preferredmolecular weight is 7-14 kDa.

The polymer, e.g., PEG, can be linked to the Sp35 polypeptide throughany suitable, exposed reactive group on the polypeptide. The exposedreactive group(s) can be, for example, an N-terminal amino group or theepsilon amino group of an internal lysine residue, or both. An activatedpolymer can react and covalently link at any free amino group on theSp35 polypeptide. Free carboxylic groups, suitably activated carbonylgroups, hydroxyl, guanidyl, imidazole, oxidized carbohydrate moietiesand mercapto groups of the Sp35 (if available) also can be used asreactive groups for polymer attachment.

Preferably, in a conjugation reaction, from about 1.0 to about 10 molesof activated polymer per mole of polypeptide, depending on polypeptideconcentration, is employed. Usually, the ratio chosen represents abalance between maximizing the reaction while minimizing side reactions(often non-specific) that can impair the desired pharmacologicalactivity of the Sp35 moiety. Preferably, at least 50% of the biologicalactivity (as demonstrated, e.g., in any of the assays described hereinor known in the art) of the Sp35 polypeptide is retained, and mostpreferably nearly 100% is retained.

The polymer can be conjugated to the Sp35 polypeptide using conventionalchemistry. For example, a polyalkylene glycol moiety can be coupled to alysine epsilon amino group of the Sp35 polypeptide. Linkage to thelysine side chain can be performed with an N-hydroxylsuccinimide (NHS)active ester such as PEG succinimidyl succinate (SS-PEG) andsuccinimidyl propionate (SPA-PEG). Suitable polyalkylene glycol moietiesinclude, e.g. carboxymethyl-NHS, norleucine-NHS, SC-PEG, tresylate,aldehyde, epoxide, carbonylimidazole, and PNP carbonate. These reagentsare commercially available. Additional amine reactive PEG linkers can besubstituted for the succinimidyl moiety. These include, e.g.,isothiocyanates, nitrophenylcarbonates, epoxides, and benzotriazolecarbonates. Conditions preferably are chosen to maximize the selectivityand extent or reaction. Such optimization of reaction conditions iswithin ordinary skill in the art.

PEGylation can be carried out by any of the PEGylation reactions knownin the art. See, e.g., Focus on Growth Factors, 3: 4-10, 1992; publishedEuropean patent applications EP 0 154 316 and EP 0 401 384. PEGylationmay be carried out using an acylation reaction or an alkylation reactionwith a reactive polyethylene glycol molecule (or an analogous reactivewater-soluble polymer).

PEGylation by acylation generally involves reacting an active esterderivative of polyethylene glycol. Any reactive PEG molecule can beemployed in the PEGylation. A preferred activated PEG ester is PEGesterified to N-hydroxysuccinimide (NHS). As used herein, “acylation”includes without limitation the following types of linkages between thetherapeutic protein and a water soluble polymer such as PEG: amide,carbamate, urethane, and the like. See, Bioconjugate Chem. 5: 133-140,1994. Reaction parameters should be chosen to avoid temperature,solvent, and pH conditions that would damage or inactivate the Sp35polypeptide.

Preferably, the connecting linkage is an amide. Preferably, at least 95%of the resulting product is mono, di- or tri-PEGylated. However, somespecies with higher degrees of PEGylation may be formed in amountsdepending on the specific reaction conditions used. Optionally, purifiedPEGylated species are separated from the mixture, particularly unreactedspecies, by conventional purification methods, including, e.g.,dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gelfiltration chromatography, and electrophoresis.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with Sp35 in the presence of a reducing agent. Inaddition, one can manipulate the reaction conditions to favor PEGylationsubstantially only at the N-terminal amino group of Sp35 (i.e., amono-PEGylated protein). In either case of mono-PEGylation orpoly-PEGylation, the PEG groups are preferably attached to the proteinvia a —CH2-NH— group. With particular reference to the —CH2- group, thistype of linkage is known as an “allyl” linkage.

Derivatization via reductive alkylation to produce a mono-PEGylatedproduct exploits differential reactivity of different types of primaryamino groups (lysine versus the N-terminal) available forderivatization. The reaction is performed at a pH that allows one totake advantage of the pKa differences between the epsilon-amino groupsof the lysine residues and that of the N-terminal amino group of theprotein. By such selective derivatization, attachment of a water solublepolymer that contains a reactive group such as an aldehyde, to a proteinis controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs.

The polymer molecules used in both the acylation and alkylationapproaches are selected from among water soluble polymers. The polymerselected should be modified to have a single reactive group, such as anactive ester for acylation or an aldehyde for alkylation, preferably, sothat the degree of polymerization may be controlled as provided for inthe present methods. An exemplary reactive PEG aldehyde is polyethyleneglycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy oraryloxy derivatives thereof (see, U.S. Pat. No. 5,252,714). The polymermay be branched or unbranched. For the acylation reactions, thepolymer(s) selected should have a single reactive ester group. Forreductive alkylation, the polymer(s) selected should have a singlereactive aldehyde group. Generally, the water soluble polymer will notbe selected from naturally-occurring glycosyl residues since these areusually made more conveniently by mammalian recombinant expressionsystems.

Methods for preparing a PEGylated Sp35 generally includes the steps of(a) reacting a Sp35 protein or polypeptide with polyethylene glycol(such as a reactive ester or aldehyde derivative of PEG) underconditions whereby the molecule becomes attached to one or more PEGgroups, and (b) obtaining the reaction product(s). In general, theoptimal reaction conditions for the acylation reactions will bedetermined case by case based on known parameters and the desiredresult. For example, the larger the ratio of PEG:protein, the greaterthe percentage of poly-PEGylated product.

Reductive alkylation to produce a substantially homogeneous populationof mono-polymer/Sp35 generally includes the steps of: (a) reacting aSp35 protein or polypeptide with a reactive PEG molecule under reductivealkylation conditions, at a pH suitable to pen-nit selectivemodification of the N-terminal amino group of Sp35; and (b) obtainingthe reaction product(s).

For a substantially homogeneous population of mono-polymer/Sp35, thereductive alkylation reaction conditions are those that permit theselective attachment of the water soluble polymer moiety to theN-terminus of Sp35. Such reaction conditions generally provide for pKadifferences between the lysine side chain amino groups and theN-terminal amino group. For purposes of the present invention, thepreferred pH is in the range of 3-9, preferably 3-6.

Sp35 polypeptides can include a tag, e.g., a moiety that can besubsequently released by proteolysis. Thus, the lysine moiety can beselectively modified by first reacting a His-tag modified with a lowmolecular weight linker such as Traut's reagent (Pierce) which willreact with both the lysine and N-terminus, and then releasing the histag. The polypeptide will then contain a free SH group that can beselectively modified with a PEG .containing a thiol reactive head groupsuch as a maleimide group, a vinylsulfone group, a haloacetate group, ora free or protected SH.

Traut's reagent can be replaced with any linker that will set up aspecific site for PEG attachment. For example, Traut's reagent can bereplaced with SPDP, SMPT, SATA, or SATP (Pierce). Similarly one couldreact the protein with an amine reactive linker that inserts a maleimide(for example SMCC, AMAS, BMPS, MBS, EMCS, SMPB, SMPH, KMUS, or GMBS), ahaloacetate group (SBAP, SIA, SIAB), or a vinylsulfone group and reactthe resulting product with a PEG that contains a free SH.

In some embodiments, the polyalkylene glycol moiety is coupled to acysteine group of the Sp35 polypeptide. Coupling can be effected using,e.g., a maleimide group, a vinylsulfone group, a haloacetate group, or athiol group.

Optionally, the Sp35 polypeptide is conjugated to the polyethyleneglycol moiety through a labile bond. The labile bond can be cleaved in,e.g., biochemical hydrolysis, proteolysis, or sulfhydryl cleavage. Forexample, the bond can be cleaved under in vivo (physiological)conditions.

The reactions may take place by any suitable method used for reactingbiologically active materials with inert polymers, preferably at aboutpH 5-8, e.g., pH 5, 6, 7, or 8, if the reactive groups are on the alphaamino group at the N-terminus. Generally the process involves preparingan activated polymer and thereafter reacting the protein with theactivated polymer to produce the soluble protein suitable forformulation.

Vectors

The invention provides vectors comprising the nucleic acids encodingSp35 polypeptides. The choice of vector and expression control sequencesto which the nucleic acids of this invention is operably linked dependson the functional properties desired, e.g., protein expression, and thehost cell to be transformed.

Expression control elements useful for regulating the expression of anoperably linked coding sequence are known in the art. Examples include,but are not limited to, inducible promoters, constitutive promoters,secretion signals, and other regulatory elements. When an induciblepromoter is used, it can be controlled, e.g., by a change in nutrientstatus, or a change in temperature, in the host cell medium.

The vector can include a prokaryotic replicon, i.e., a DNA sequencehaving the ability to direct autonomous replication and maintenance ofthe recombinant DNA molecule extra-chromosomally in a bacterial hostcell. Such replicons are well known in the art. In addition, vectorsthat include a prokaryotic replicon may also include a gene whoseexpression confers a detectable marker such as a drug resistance.Examples of bacterial drug resistance genes are those that conferresistance to ampicillin or tetracycline.

Vectors that include a prokaryotic replicon can also include aprokaryotic or bacteriophage promoter for directing expression of thecoding gene sequences in a bacterial host cell. Promoter sequencescompatible with bacterial hosts are typically provided in plasmidvectors containing convenient restriction sites for insertion of a DNAsegment to be expressed. Examples of such plasmid vectors are pUC8,pUC9, pBR322 and pBR329 (BioRad), pPL and pKK223 (Pharmacia). Anysuitable prokaryotic host can be used to express a recombinant DNAmolecule encoding a protein of the invention.

Eukaryotic cell expression vectors are known in the art and arecommercially available. Typically, such vectors contain convenientrestriction sites for insertion of the desired DNA segment. Exemplaryvectors include pSVL and pKSV-10 (Pharmacia), pBPV-1, pML2d(International Biotechnologies), pTDT1 (ATCC 31255), retroviralexpression vector pMIG, adenovirus shuttle vector pDC315, and AAVvectors.

Eukaryotic cell expression vectors may include a selectable marker,e.g., a drug resistance gene. The neomycin phosphotransferase (neo) gene(Southern et al., 1982, J. Mol. Anal. Genet. 1:327-341) is an example ofsuch a gene.

To express the antibodies or antibody fragments, DNAs encoding partialor full-length light and heavy chains are inserted into expressionvectors such as plasmids, retroviruses, cosmids, YACs, EBV-derivedepisomes, and the like. The expression vector and expression controlsequences are chosen to be compatible with the expression host cellused. The antibody light chain gene and the antibody heavy chain genecan be inserted into separate vector. In some embodiments, both genesare inserted into the same expression vector.

A convenient vector is one that encodes a functionally complete humanC_(H) or C_(L) immunoglobulin sequence. Preferably, restriction sitesengineered so that any V_(H) or V_(L) sequence can be easily insertedand expressed. In such vectors, splicing usually occurs between thesplice donor site in the inserted J region and the splice acceptor sitepreceding the human C region, and also at the splice regions that occurwithin the human C_(H) exons. Polyadenylation and transcriptiontermination occur at native chromosomal sites downstream of the codingregions. The recombinant expression vector can also encode a signalpeptide that facilitates secretion of the antibody chain from a hostcell.

Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and enhancers derived from retroviralLTRs, cytomegalovirus (CMV) (such as the CMV promoter/enhancer), SimianVirus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g.,the adenovirus major late promoter (AdMLP)), polyoma and strongmammalian promoters such as native immunoglobulin and actin promoters.For further description of viral regulatory elements, and sequencesthereof, see e.g., Stinski U.S. Pat. No. 5,168,062; Bell U.S. Pat. No.4,510,245; and Schaffner U.S. Pat. No. 4,968,615.

The recombinant expression vectors may carry sequences that regulatereplication of the vector in host cells (e.g., origins of replication)and selectable marker genes. The selectable marker gene facilitatesselection of host cells into which the vector has been introduced (seee.g., Axel U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017). Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Preferred selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr-hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection).

Nucleic acid molecules encoding Sp35 polypeptides and anti-Sp35antibodies, and vectors comprising these nucleic acid molecules, can beused for transformation of a suitable host cell. Transformation can beby any suitable method. Methods for introduction of exogenous DNA intomammalian cells are well known in the art and include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene-mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei. In addition, nucleic acid molecules may be introduced intomammalian cells by viral vectors.

Transformation of host cells can be accomplished by conventional methodssuited to the vector and host cell employed. For transformation ofprokaryotic host cells, electroporation and salt treatment methods canbe employed (Cohen et al., 1972, Proc. Natl. Acad. Sci. USA69:2110-2114). For transformation of vertebrate cells, electroporation,cationic lipid or salt treatment methods can be employed. See, e.g.,Graham et al., 1973, Virology 52:456-467; Wigler et al., 1979, Proc.Natl. Acad. Sci. USA 76:1373-1376f.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC). These include, inter alia, Chinesehamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamsterkidney (BHK) cells, monkey kidney cells (COS), human hepatocellularcarcinoma cells (e.g., Hep G2), A549 cells, and a number of other celllines.

Expression of polypeptides from production cell lines can be enhancedusing known techniques. For example, the glutamine synthetase (GS)system is commonly used for enhancing expression under certainconditions. See, e.g., European Patent Nos. 0216846, 0256055, and0323997 and European Patent Application No. 89303964.4.

Host Cells

Host cells can be prokaryotic or eukaryotic. Preferred eukaryotic hostcells include, but are not limited to, yeast and mammalian cells, e.g.,Chinese hamster ovary (CHO) cells (ATCC Accession No. CCL61), NIH Swissmouse embryo cells NIH-3T3 (ATCC Accession No. CRL1658), and babyhamster kidney cells (BHK). Other useful eukaryotic host cells includeinsect cells and plant cells. Exemplary prokaryotic host cells are E.coli and Streptomyces.

Formulations

Compositions containing Sp35 polypeptides, anti-Sp35 antibodies, orantigen binding fragments of anti-Sp35 antibodies may contain suitablepharmaceutically acceptable carriers. For example, they may containexcipients and/or auxiliaries that facilitate processing of the activecompounds into preparations designed for delivery to the site of action.Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substances thatincrease the viscosity of the suspension include, for example, sodiumcarboxymethyl cellulose, sorbitol and dextran. Optionally, thesuspension may also contain stabilizers. Liposomes also can be used toencapsulate the molecules of the invention for delivery into cells orinterstitial spaces. Exemplary pharmaceutically acceptable carriers arephysiologically compatible solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, water, saline, phosphate buffered saline, dextrose, glycerol,ethanol and the like. In some embodiments, the composition comprisesisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride. In some embodiments, the compositionscomprise pharmaceutically acceptable substances such as wetting or minoramounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the active ingredients.

Compositions of the invention may be in a variety of forms, including,for example, liquid (e.g., injectable and infusible solutions),dispersions, suspensions, semi-solid and solid dosage forms. Thepreferred form depends on the mode of administration and therapeuticapplication.

The composition can be formulated as a solution, micro emulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared byincorporating the active ingredient in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active ingredient into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution. Theproper fluidity of a solution can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The active ingredient can be formulated with a controlled-releaseformulation or device. Examples of such formulations and devices includeimplants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, for example, ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for the preparation ofsuch formulations and devices are known in the art. See e.g., Sustainedand Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,Marcel Dekker, Inc., New York, 1978.

Injectable depot formulations can be made by forming microencapsulatedmatrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the polymer employed, the rate of drug release can becontrolled. Other exemplary biodegradable polymers are polyorthoestersand polyanhydrides. Depot injectable formulations also can be preparedby entrapping the drug in liposomes or micro emulsions.

Supplementary active compounds can be incorporated into thecompositions. In some embodiments, an Sp35 polypeptide, anti-Sp35antibody or fragment thereof is coadministered with an anti-NgR1antibody, or an antigen-binding fragments thereof, or soluble NgR1polypeptides or NgR1 fusion protein.

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time, or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. It is advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.See, e.g., Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton,Pa. 1980).

In addition to the active compound, the liquid dosage form may containinert ingredients such as water, ethyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan.

Gene Therapy

An Sp35 polypeptide can be produced in vivo in a mammal, e.g., a humanpatient, using a gene therapy approach to treatment of a CNS disease,disorder or injury in which reducing inhibition of axonal extensionwould be therapeutically beneficial. This involves administration of asuitable Sp35 polypeptide-encoding nucleic acid operably linked tosuitable expression control sequences. Preferably, these sequences areincorporated into a viral vector. Suitable viral vectors for such genetherapy include adenoviral vectors, lentiviral vectors, baculoviralvectors, Epstein Barr viral vectors, papovaviral vectors, vaccinia viralvectors, herpes simplex viral vectors, and adeno associated virus (AAV)vectors. The viral vector can be a replication-defective viral vector. Apreferred adenoviral vector has a deletion in its E1 gene or E3 gene.When an adenoviral vector is used, preferably the mammal is not exposedto a nucleic acid encoding a selectable marker gene.

EXAMPLES

The invention is further illustrated by the following experimentalexamples. The examples are provided for illustrative purposes only, andare not to be construed as limiting the scope or content of theinvention in any way.

Example 1 Sp35 Expression Pattern

Expression of Sp35 in human tissues was evaluated by Northern blotanalysis. Multiple tissue blots containing 12 human major tissues or 14human CNS tissues were hybridized overnight at 68° C. with P³² labeledSp35 probe (nucleotides 150-450 of the Sp35 cDNA sequence). The blotswere washed 3 times with 2×SSC, 0.5% SDS, then 3 times with 0.5×SSC,0.1% SDS. The blot was then exposed to X-ray film and the mRNA levelsvisualized by autoradiography.

Sp35 was highly expressed in the human brain but not in heart, skeletalmuscle, colon, thymus, spleen, kidney, liver, small intestine, placenta,lung and peripheral blood leukocytes. Sp35 was expressed in all braintissues tested, including tissues isolated from frontal cortex,posterior cortex, entorhinal cortex, hippocampus, olfactory bulb,striatum, thalamus, cerebellum, midbrains, pons, medulla and spinalcord. A gradient of gene expression along the rostral/cordal axis wasobserved for Sp35, with highest levels in the cortical cortex and lowestlevels in the spinal cord.

Immunohistochemical (IHC) staining was used to determine if Sp35 isexpressed in specific brain cells. 4% paraformaldehyde fixed rat brains,spinal cord sections, or primary granular neuron cultures were incubatedwith primary antibodies to Sp35, as indicated, followed by secondaryantibodies conjugated to Alexa 480 or 590 (Molecular Probes Inc.). Thesections were then mounted in Vectashield and visualized by fluorescencemicroscopy. The anti-Sp35 specific antibodies used for IHC weregenerated from a Fab phage display library using MorPhosys technology.

Sp35 is expressed specifically in neurons and oligodendrocytes, but notin astrocytes. This was determined in experiments in which Rat braintissue sections were stained with various agents, includinganti-astrocyte marker GFAP, an antibody to an oligodendrocyte marker(O4), and an antibody to the neuronal marker βIII tubulin, allcounterstained with an anti-Sp35 antibody. Oligodendrocytes and neuronswere intensely stained with the anti-Sp35 antibody. No staining ofastrocytes was observed.

As an independent confirmation of the expression pattern of Sp35, weperformed semi quantitative RT-PCR using mRNA extracted (Ambion kit)from rat primary cell cultures of purified astrocytes, oligodendrocytes,and cerebellum granular neurons. Forward primer AAGGCCCAGCAGGTGTTTGTGGA(SEQ ID NO: 14) and reverse primer TACTCGATCTCGATGTTGTGCTTT (SEQ ID NO:15) were used. Following 26 cycles, a strong band was observed in themRNA from neurons, a distinct but weaker signal was detected inoligodendrocyte mRNA, and no band was observed in astrocytes.

Example 2 Sp35-Fc Fusion Protein

To study the biological function of Sp35, a construct was made fusingthe extra-cellular portion of human Sp35 (residuse 1-531) to the hingeand Fc region of human IgG1. A partial coding sequence for human Sp35was obtained by PCR from clone 227.2 (Incyte) using the forward primer5′CAGCAGGTCGACGCGGC CGCATGCTGGCGGGGGGCGT3′ (SEQ ID NO: 16) and reverseprimer 5′CAGCAGGTCGACCTCGCCCGGCTGGTTGG3′ (SEQ ID NO: 17).

The blunt end PCR product was subcloned into the SrfI site of the PCRSCRIPT AMP vector (Stratagene) to create PCR SCRIPT AMP-sp35. A SalIfragment was isolated from PCR SCRIPT AMP-sp35 and subcloned into thePCRCAMP Ig vector (derivative of Stratagene vector PCR SCRIPT AMPwherein the Fc gamma sequence is subcloned as a SalI(5′) to NotI(3′)fragment), fusing the Sp35 signal sequence and ectodomain sequence(codons 1-531) in-frame with sequences encoding the hinge and Fc regionof human IG1. Correct isolates were identified, and a NotI fragmentencompassing the Sp35 Fc fragment was subcloned into the single NotIcloning site of the 293E expression vector, CH274, a derivative ofcommercial expression vector REP4 (Invitrogen). The Sp35-Fc fusionencoded by the new vector, CH274/sp35-Fc , was confirmed by DNAsequencing as plasmid GT123.

Stable cell lines expressing Sp35-Fc fusion protein were generated byelectroporation of CHO host cells DG44 with plasmid GT123. TransfectedCHO cells were cultured in alpha minus MEM in the presence of 10%dialyzed serum and 4mM glutamine to select for nucleoside-independentgrowth. Fourteen days post-transfection, cells were fed fresh media. Toscreen for cells expressing Sp35-Fc, CHO cells were labeled withPhycoerythrin (PE)-labeled goat anti-human IgG (Jackson Labs) andsubjected to high speed flow cytometry sorting in a FACS Mo-Flo(Cytomation). The cells that expressed the highest levels of Sp35-Igwere selected. These cells were expanded in culture for 7 days, thenre-labeled and re-sorted. Cells expressing the highest levels of Sp35-Igwere isolated as individual clones in 96-well plates. These clones weregrown for two weeks and then fed fresh media one day prior to FACSanalysis to check for expression levels. Clones that expressed thehighest levels of Sp35-Fc were expanded, and frozen cell banks wereestablished. The cell lines were adapted to grow in suspension culturein the serum free media BCM16. The titer of Sp35-Fc produced by theseclones was determined by growing cell lines at 37° C. for 4-5 passages,then growing the cells to 50% maximal cell density and culturing themfor 10-15days at 28° C. until the viable cell density dropped to 75%. Atthis time, the culture media were harvested, cleared of cells and debrisby centrifugation, and the culture supernatants titered for Sp35-Fclevels by Western blot analysis using an anti-human Ig antibody (JacksonLab) as the probe.

Sp35-Fc fusion protein was purified from the clarified culture medium asfollows: 9 ml of 1M HEPES pH 7.5 was added to 900 ml of conditionedmedium. The medium was batch loaded for 3 hr at 4° C. onto 3 ml ofProtein A Sepharose (Pharmacia). The resin was collected in a 1.5 cm(I.D.) column, and washed four times with 3 ml PBS, two times with 4 mlof PBS containing 800 mM NaCl, and then again with 3 mL of PBS. TheSp35-Fc was eluted from the column with 25 mM NaH₂PO₄ pH 2.8, 100 mMNaCl in 1.5 mL fractions and neutralized by adding 75 μL of 0.5 MNaH₂PO₄ pH 8.6. Peak protein-containing fractions were identified byabsorbence at 280 nm, pooled, and subjected to further purification on a1 mL Protein A column. Prior to loading, NaCl was added to 600 mM andHEPES pH 7.5 to 50 mM. The column was washed twice with 600 μL of 10 mMHEPES pH 7.5, 1 M NaCl, and then with 1 mL PBS. Sp35-Fc was eluted fromthe column with 25 mM NaH₂PO₄ pH 2.8, 100 mM NaCl, collecting 0.5 mLfractions, and neutralized by adding 25 μL of 0.5 M NaH₂PO₄ pH 8.6. Peakprotein-containing fractions were identified by absorbance at 280 nm andpooled. By reducing SDS-PAGE, the Sp35-Ig migrated as a single band(>95% pure) with an apparent mass of 90 kDa. Under non-reducingconditions, the protein ran as a dimer with an approximate mass of 180kDa. The purified Sp35-Fc was aliquoted and stored at −70° C. The NotIfragment of GT123, which contains Sp35 amino acids 1-531 and human IgG1Fc, was subcloned into the PV90 vector NotI site to create DB002.

Example 3 His-AP-Sp35 Fusion Protein

To study and isolate the receptor for Sp35, the protein was expressed inCOS7 and CHO cells as a His-tagged-alkaline phosphatase (His-AP) fusionprotein. The plasmid was constructed as follows: The extracellulardomain of Sp35 (a.a. 34-532) was PCR amplified using primers (forward)5′-AATTAAGAATTCACGGGCTGCCCGCCCCGCTGCGAGT-3′ (SEQ ID NO: 18), containingan Eco RI cleavage site (underlined), and (reverse)5′-TATATTTCTAGATCACTCGCCCGGCTGGTTGGAGATGAAAGCGA-3′ (SEQ ID NO: 19),containing an Xba I cleavage site (underlined). The PCR product wascleaved with Xba I, the resulting sticky-end filled in with T4 DNApolymerase, then digested with Eco RI and gel purified. The digestedproduct was ligated into a Hind III-filled in/EcoRI His-AP fragment fromthe His-AP-pcDNA 1.1 vector (Invitrogen). The His-AP-Sp35 fragment wasdigested with Hind III and Eco RI, filled in, then ligated into theNotI-filled site in vector pV90. The DNA sequence of the insert wasconfirmed by DNA sequencing.

COS7 cells were split the day before transfection. His-AP-Sp35 vectorDNA (8 μg) was used to transfect 5×10⁶ cells using lipofectamine(Invitrogen). The conditioned medium was harvested 48 hr posttransfection.

We developed a CHO cell line expressing the His-AP-Sp35 fusion proteinusing the pV90 plasmid. CHO host cells DG44 (2×10⁶ cells) weretransfected with 100 μg of plasmid by electroporation. Cells werecultured in alpha minus MEM in the presence of 10% dialyzed serum and 4mM glutamine to select for nucleoside-independent growth. Fourteen dayspost transfection cells were fed fresh media in anticipation ofscreening by FACS Mo-Flo (Cytomation) sorting. Transfected CHO cellswere labeled with the mouse monoclonal antibody 8B6 directed againsthuman placental alkaline phosphatase (Sigma). A secondary antibody,PE-labeled goat anti-mouse IgG, was used to produce a signal specificfor transfected cells. After PE-labeling, cells were subjected to highspeed flow cytometry sorting and the top 5% selected.

To produce conditioned medium with His-AP-Sp35, the cells that expressedthe highest levels of HIS-ApSp35 were selected. The cell lines wereadapted to grow in suspension culture in serum free media (BCM16). Thetiter of His-AP-Sp35 that was produced by these clones was determined bygrowing cell lines at 37° C. for 4-5 passages, then growing the cells to50% of maximal cell density and culturing them for 10-15 days at 28° C.until the viable cell density dropped to 75%. The culture media wereharvested, cleared of cells and debris by centrifugation, and theculture supernatants titered for His-AP-Sp35 levels by western blotanalysis using anti-human AP antibody (Jackson Labs) as the probe.

His-AP-Sp35 was purified from the conditioned medium as follows: 400 mLof conditioned medium from CHO cells expressing His-AP-Sp35 was dilutedwith 400 mL of water. Triethanolamine pH 8.5 was added to 25 mM from a0.5 M stock and the sample was batch loaded for 2 hours at 4° C. onto 6ml of Fractogel TMAE (EM Industries) anion exchange resin. The resin wascollected in a 1.5 cm (I. D.) column, and washed two times with 6 mL of10 mM HEPES pH7.5, 50 mM NaCl. The AP-Sp35 was eluted from the columnwith 10 mM HEPES pH 7.5, 200 mM NaCl in 2 mL fractions. Peak fractionswere identified by monitoring AP activity and by SDS-PAGE. Theflow-through fraction from the TMAE column was further diluted with 300ml of water and loaded in batch overnight at 4° C. onto 6 ml of TMAEresin. The resin was collected and washed as described above and elutedwith 10 mM HEPES pH 7.5, 150 mM NaCl. Peak fractions again wereidentified by monitoring AP activity and by SDS-PAGE. His-AP-Sp35 fromthe first column was 50% pure and AP-Sp35 from the second column was 90%pure. Under reducing conditions, the His-AP-Sp35 migrated on SDS-PAGEgels with an apparent mass of 130 kDa. While the 90% pure material wasappropriate for most investigations, for some studies the His-AP-Sp35was further purified on Ni-NTA agarose resin (Qiagen). NaCl was added tothe elution fractions from the TMAE column to 800 mM and 0.5 Mtriethanolamine pH. 8.5 and 1M imidazole pH 7.0 was added to 25 mM and15 mM, respectively. 4.5 ml of the sample was loaded onto a 400 μL NiNTAcolumn. The column was washed three times with 25 mM Triethanolamine pH8.5, 800 mM NaCl, 15 mM imidazole, and the His-AP-Sp35 was eluted fromthe column with 200 mM imidazole pH 7.0, 350 mM NaCl, collecting 200 μLfractions. Peak AP-containing fractions were pooled and dialyzedovernight against 250 volumes of 10 mM HEPES pH 7.5, 200 mM NaCl. MgCl₂and ZnCl₂ were added to the retentive to 2 and 0.25 mM, respectively.The final product was greater than 95% pure by SDS-PAGE, and ran as asingle band with mass of approximately 140 kDa under reducingconditions.

The Sp35 constructs were also engineered as Fc fusions. An Sp-35 LRR-Fcconstruct was generated by PCR using primers (forward) 5′CTTGACACGGGATCCGCGGCCGCATGCTGGCGGGGGGCGTGAGG3′ (SEQ ID NO: 20) and (reverse)5′GCAGCGGGGCGGGCAGCCCGTGGCCGAGCCTGACAGC ACTGAGCC3′ (SEQ ID NO: 21). ThePCR product was inserted into the NotI site of PV90 vector. Sp35 IG-FCconstruct was generated by PCR using primers (forward)5′CTTGACACGGGATCCGCGGCCGCATGCTGGCGGGGGGC GTGAGG3′ (SEQ ID NO: 22) and(reverse) 5′GTCCCGGATGCGGGCGCGGG CCGAGCCTGACAGCACTGAGCCCAG3′ (SEQ ID NO:23). The PCR product was inserted into the NotI site of PV90 vector. Theproteins were expressed in CHO cells and purified using a protein Asepharose column.

Example 4 Sp35 Binding to NgR1-Expressing Cells

Four different methods were used to show Sp35 binding to NgR1. First wedetected the interaction in a direct binding assay in which the alkalinephosphatase-Sp35 conjugate (AP-Sp35) was incubated with NgR1 expressingcells and binding assessed using a chromogenic AP detection reagent. 90%confluent COS7 cells were grown on 100 mm tissue culture dishes andtransfected with NgR1 expressing plasmids using Fugene 6 reagents(Roche). After 48 hours, the transfected cells were washed once with HBH(Hank's balanced salt buffer, 1 mg/ml BSA, 20 mM HEPES, pH 7.0), andthen incubated for 1.5 hr at 23° C. with 4 μg/ml of the AP-Sp35 fusionprotein in HBH. The cells were washed with ice-cold HBH buffer 3 timesfor 3 min each, then fixed with 3.7% formaldehyde in 20 mM HEPES, pH7.0, 150 mM NaCl for 15 min, and transferred back into HBH buffer.Endogenous heat-labile AP was heat-inactivated for 2 hours at 67° C.Bound AP-Sp35 was detected by incubation with nitro blue tetrazolium NBT(Roche). Ap-Sp35 bound to COS7 cells expressing human NgR1 receptor, butnot to control COS7 cells transfected with the vector alone. A punctatestaining pattern for NgR1 was observed, reflecting that only a fraction,probably 50% of the cells, were transfected with the NgR1.

To better quantify binding, we performed the same experiment butparallel cell samples were treated with 8, 4, 2, 1, 0.5, 0.125, 0.06μg/ml of the AP-Sp35. Bound AP was incubated with 4-nitrophenylphosphate and AP activity assessed in a 96-well plate reader (MolecularDevices). From these data, we estimated that the EC50 for binding ofAp-Sp35 to human NgR1 was approximately 6 nM.

Second, we detected binding of Sp35 to NgR1 in an ELISA approach. ELISAplates (Costar) were coated with 10 μg/ml soluble NgR1-Fc receptors(sNgR310-Fc containing rat NgR1 peptide 35-310 fused to the hinge and Fcof rat IgG1 and sNgR344-Fc containing rat NgR1 peptide 35-344 fused tothe rat IgG1) in 0.1M NaHCO₃, pH 9.0 for 1 hr at 37° C. The plates wereblocked and washed with 25 mM Hepes, pH 7.0, 0.1% BSA, 0.1% ovalbumin,0.1% non-fat dried milk and 0.001% NaN₃. AP-Sp35 protein, 4 μg/ml, wasadded to the plate and incubated overnight at 4° C. The plates were thenwashed with 10 mM Tris pH 7.5, 150 mM NaCl and bound AP detected using10 μg/ml of the chromogenic substrate 4-nitrophenyl phosphate diluted in0.1 M glycine, 1 mM MgCl₂, 1 mM ZnCl₂ pH 10.5. The OD₄₁₀ was determinedin an ELISA reader (Molecular Devices) equipped with the Softmaxprogram. AP-Sp35 bound to immobilized sNgR-344-Fc but not thesNgR-310-Fc protein, indicating that the longer version of NgR1 wasrequired for Sp35 binding. We were able to compete the binding ofAP-Sp35 to sNgR344-Fc NgR1 by 80% by pre-incubating the AP Sp35 with100-fold excess of sNgR344-Fc. No competition of binding was seen usinga hedgehog-rat Ig1 fusion protein as a rat Ig fusion control protein.

Third, we detected the binding of Sp35 to NgR1 by co-immunoprecipitatingSp35 with NgR1. For this study, 80% confluent COS7 cells, grown on 100mm tissue culture dishes, were transfected with plasmids encodingSp35-hemagglutinin (Sp35-HA) and NgR-FLAG using Fugene 6 reagents(Roche) 48 hours after transfection, cells were harvested and lysed in 1ml lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mMEGTA, 1% Triton X-100 and 10% glycerol) at 4° C. for 30 min. The lysatewas then centrifuged at 14,000×g for 15 min, and the supernatantscollected and incubated 4° C. overnight with agitation, using anti-HAaffinity matrix (Roche). The samples were washed 3 times with 1 ml oflysis buffer, then boiled for 3 minutes in Laemmli sample buffer,subjected to 4-15% SDS-PAGE, and analyzed by immuno-blotting withAnti-FLAG M2 antibody (Sigma). The anti-HA tag affinity resin collecteda complex containing both Sp35-HA and FLAG-NgR, as is evident by thepresence of FLAG. This complex was not seen in lysates from controltransfections in which cells were treated with Sp35-HA plasmid orFLAG-NgR1 plasmid alone, or cells that were co-transfected with theFlag-NgR1 and a HA-tagged control protein that does not bind NgR1.

Sp35-HA was made as follows. The Sp35 signal sequence and extracellulardomain (amino acids 1-531) was PCR amplified using primers5′ATATTCTAGAATGCTGGCGGGGGGCGTGAG3′ (SEQ ID NO: 24) and5′ATATACTAGTGTCGTTGCCGCCCGCGTTGG3′ (SEQ ID NO: 25) containing XbaI andSpeI sites (underlined). The PCR product was digested by XbaI and SpeIand inserted into the vector pCGCHA between the Xba I and Spe I sites.The sequence of the insert was confirmed by DNA sequencing. The FLAGNgR1 construct was gift from Dr. Zhigang He (Nature, Vol 420, Nov. 7,2002).

Fourth, we showed that Ap-Sp35 bound to rat cerebellum granular neurons(CGN) which express NgR1. For this experiment, 90% confluent postnatalday 8 CGN cells were grown on 100 mm tissue culture dishes. After 48hours, the cells were washed once with HBH buffer, and then incubatedwith 4 μg/ml of AP-Sp35 in HBH buffer for 1.5 hour at 23° C. The cellswere then washed with ice-cold HBH 3 times for 3 minutes each, thenfixed with 3.7% formaldehyde in 20 mM HEPES, pH 7.0, and 150 mM NaCl for15 min, and transferred back to HBH. Endogenous heat labile AP washeat-inactivated for 2 hours at 67° C. Bound AP-Sp35 was detected byincubation with nitro blue tetrazolium NBT (Roche). AP-Sp35 bound topostnatal day 8 cerebellum granular neurons, which express NgR1. Thebinding of AP-Sp35 to the neurons was inhibited by treating the CGN withPIPLC (5 units/ml) which cleaves most GPI anchored proteins frommembrane surfaces. Since NgR1 is a GPI-linked protein, this resultfurther supports the notion that Sp35 is binding to NgR1 on CGN cells.

Example 5 Co-localization of Sp35 with NgR1

To determine whether Sp35 and NgR1 are expressed in the same neurons, weperformed a co-localization study. 4% paraformaldehyde-fixed rat p8primary granular neuron cultures were incubated with antibodies againstSp35 and NgR1(Santa Cruz), and then with the appropriate Alexa-labeledsecondary antibodies (Molecular Probes Inc.). The cells were visualizedby con-focal fluorescence microscopy. Neurons were intensely stained bythe Sp35 and NgR1 antibodies. Both proteins were expressed in the cellbodies and axons of neurons. To aid in the co-localization analysis,different colored probes were used for the 2 types of antibodies. Whenthe stains (red for NgR positive cells and green for Sp35 positivecells) were merged we saw a yellow color throughout the cell indicatingthat the two proteins were co-localized within the neurons.

Example 6 NgR1 Binding Sites within Sp35

We used deletion mapping to define the specific domains of Sp35 involvedin NgR1 interactions. The following deletion constructs were made usingthe Stratagene Quikchange Mutagenesis kit. We verified all vectorconstructs by DNA sequencing of the modified inserts.

His-AP-Sp35b which contains the leucine rich repeat domain of Sp35 plusthe basic region (a.a 34-432) was cloned from the His-AP-Sp35 (a.a.34-532) vector by PCR. Primers used were 5′CCCAGCAGGTGTTTGTGGACGAGTGATCTAGGGCCGCGGATCCCTG-3′ (SEQ ID NO: 26) and 5′-CAGGGATCCG CGGCCCTAGATCACTCGTCCACAAACACCTGCTGGG-3′ (SEQ ID NO: 27).

His-AP-Sp35d, which encodes the Ig domain of Sp35 plus the basic region(a.a 417-531), was cloned from the His-AP-Sp35a (a.a. 37-531) vector byPCR. Primers used were 5′CGCCGCGCACCCGGGTGAATTCCGCGCCCGC ATCCGGGACCGC-3′(SEQ ID NO: 28) and 5′-GCGGTCCCGGATGCGGGCGC GGAATTCACCCGGGTGCGCGGCG-3′(SEQ ID NO: 29).

His-AP-Sp35e, which encodes only the Ig domain (a.a 425-531), was clonedfrom the His-AP-Sp35(aa 34-532) vector by PCR. Primers used were5′-CGCCGCGCACCCGGGTGAATTCGCCCAGCAGGTGTTTGTGGAC-3′ (SEQ ID NO: 30) and5′-GTCCACAAACACCTGCTGGGCGAATTCACCCG GGTGCGCGGCG-3′ (SEQ ID NO: 31).

A commercial mutagenesis kit and protocol (Stratagene Quikchange) wereused to mutate amino acid 456 (from arginine to glutamic acid) and aminoacid 458 (from histidine to valine) of Sp35 in the vector His-AP-Sp35(34-532). The primers used were 5′-CATCCTCTGGCTCTCACCCGAAAAGGTACTGGTCTCAGCCAAGAGC-3′ (SEQ ID NO: 32) and 5′-GCTCTTGGCTGAGACCAGTACCTTTTCGGGTGAGAGCCAGA GGATG-3′ (SEQ ID NO: 33).

His-AP-Sp35 deletion constructs (FIG. 3) were engineered in pV90expression vectors and expressed in 293 cells. The conditioned mediumwas collected and the AP adducts purified by sequential chromatographysteps on Fractogel TMAE resin and NiNTA agarose. The purified proteinswere tested for binding to NgR1 expressed on COS7 cells. The threeconstructs all bound weakly to Sp35. These results indicated that theSp35 LRR repeat 1-14 (amino acid 34 to 417) and the Ig domain of Sp35(amino acid 425-531) both contribute to Sp35 binding to NgR1. The Igdomain showed higher affinity than the LRR domain.

A structural model for the Ig domain of Sp35 was generated using theNCAM crystal structure as a framework (Rasmussen et al., 2000, Nat.Struct. Biol. 7:389-393). From this model, we observed a loop (residuenumbers 454-458, amino acids: SPRKH; SEQ ID NO. 34) that might beinvolved in binding. To test this hypothesis, we engineered an Sp35construct in which residues R at 456 and H at 458 were changed into Eand V, respectively. When this construct was tested for NgR1 binding, weobserved a >10 fold drop in signal. As an alternative approach to testthe contribution of this loop region in binding, we synthesized apeptide corresponding to the sequence LSPRKH (SEQ ID NO: 10) that wecyclized by adding cysteines at the N and C terminus of the peptide.Upon binding to NgR1, this peptide blocks, inhibits, or interferes withthe function of NgR1.

Example 7 Sp35 induces p8 CGN fasciculation

To determine the biological function of Sp35 in neurons, we incubatedSp35-Fc with postnatal day 8 granular neurons to see if Sp35 canregulate neurite out-growth. Labtek culture slides (8 well) were coatedwith 0.1 mg/ml poly-D-lysine (Sigma) before spotting of Sp35-Fc protein(16 μg/well protein). The slides were dried overnight, then rinsed andcoated with 10 μg/ml laminin (Gibco). Cerebellum granular neurons frompostnatal day 8 were dissociated and seeded onto the precoated slides.The slide cultures were incubated at 37° C. in 5% CO₂ for 24 hours. Theslides were then fixed in 4% paraformaldehyde containing 20% sucrose andstained with anti βIII tubulin (Covance TUJ1). After 24 hours, the CGNshowed clear fasciculation morphology as evident by bundling of theneurons. The fasciculation was not seen in the untreated cells or Fcprotein-coated sample controls.

Example 8 Effects of Sp35 on RhoA activation/inactivation

Sp35-Fc induced postnatal cerebellum granular neurons to undergofasciculation. Because the signaling molecule RhoA is known to beinvolved in fasciculation, we determined if Sp35-Fc can regulate RhoAfunctions in neurons. We performed the RhoA activation experiment asfollows: 293 cells or COS7 cells were transfected with expressionvectors containing combinations of RhoA, Sp35 or NgR1 using Fugene 6reagents (Roche). 48 hr post-transfection, cells were serum starvedovernight, then lysed in 50 mM Tris, pH 7.5, 1% Triton X-100, 0.5%sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl₂, plus a proteaseinhibitor cocktail. Cell lysates were clarified by centrifugation at13,000×g at 4° C. for 5 minutes, and 95% of the supernatants wereincubated with 20 μg of an immobilized GST-Rho binding domain affinitymatrix (Rhotekin beads, Upstate Biotechnology) at 4° C. for 45 minutes.The beads were washed 3 times with wash buffer (50 mM Tris, pH 7.5, 1%Triton X-100, 150 mM NaCl, 10 mM MgCl₂, with protease inhibitors).GTP-bound Rho was eluted from the beads by heating at 95° C. for 5 minin SDS-PAGE sample buffer. Bound and total Rho proteins were detected bywestern blotting using a monoclonal antibody against RhoA (Santa Cruz).COS7 and HEK293 cells transfected with Sp35 induced RhoA activation, asevident by an increase in the amount of RhoA-GTP detected in the blotfollowing transfection with the Sp35 gene. A further enhancement inRhoA-GTP was observed following treatment with Sp35-Fc. In contrast tothe increase in RhoA-GTP following transfection with Sp35 alone, whencells were transfected with Sp35 and NgR1, RhoA was partiallyinactivated. Treatment of these cells with Sp35-Fc resulted in furtherinactivation of RhoA.

We confirmed a signaling response by Sp35 using a FLIPR assay (MolecularDevices) to determine the affects of Sp35 treatment on Ca⁺⁺ flux. Weobserved a significant Ca⁺⁺ flux in cells expressing Sp35 with treatmentof Sp35-Fc, but not in the control cells treated with Sp35-Fc. The Ca⁺⁺flux was reduced when cells that had been co-transfected with NgR1 andSp35 were treated with Sp35-Fc fusion protein.

Example 9 Sp35 Protein Interaction with Itself

Because LRR domains frequently are involved in homotypic interactions,and we observed that the addition of soluble Sp35 to Sp35-transfectedcells caused an increase in RhoA-GTP over what was observed with Sp35transfections alone, we tested for Sp35 binding to itself. To performthis test, we used co-immunoprecipitation. COS7 cells 80% confluent,grown on 100 mm tissue culture dishes, were transfected with plasmidsSp35 HA or Sp35-FLAG, or both, using Fugene 6 reagents (Roche).Forty-eight hours after transfection, cells were harvested and lysed in1 ml lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mMEGTA, 1% Triton X-100 and 10% glycerol) at 4° C. for 30 min. The lysatewas then centrifuged at 14,000×g for 15 min, and the supernatantscollected and incubated, at 4° C. overnight with agitation, with ananti-HA affinity matrix (Roche). The samples were then washed 3 timeswith 1 ml of lysis buffer, boiled in Laemmli sample buffer, subjected to4-15% SDS-PAGE, and analyzed by immuno-blotting with anti-FLAGantibodies. The Anti-HA antibody resin captured a complex that containedSp35-FLAG, as determined by Western blotting. This indicated a directinteraction of Sp35 with itself. We also treated cells transfected withHA-Sp35 with Sp35-Fc and used a similar immunoprecipitation approach toshow that HA-Sp35 bound to Sp35-Fc.

Sp35-FLAG was made as follows. Sp35 gene extracellular domain (a.a.1-531) was PCR amplified using primers 5′AATTAAGCGGCCGCATGCTGGCGGGGGGCGT3′ (SEQ ID NO: 35) and 5′AATTAAGCGGCCGCTTTGTCATGT′3 (SEQ ID NO:36) containing NotI sites (underlined). The PCR product was digested byNotI and inserted into the NotI site of vector pV90. The DNA sequence ofthe insert was confirmed by DNA sequencing.

Example 10 In Vivo Transplantation of Sp35-Transformed Cells

To determine the biological function of Sp35 in spinal cord injuredrats, we infected cortical primary cultured cells (mixed cultures) withretrovirus expressing full length Sp35 or a retrovirus control, fordelivery into the injured epicenter of rat spinal cords. 2×10⁶ cellswere introduced, and the rats were sacrificed at day 10. The spinalcords were fixed in 4% paraformaldehyde overnight, then dehydrated in70%, followed by 95% ETOH. Tissue samples were imbedded in paraffin.Sections (10 microns thick) were used for immunohistochemical staining.Rats that received Sp35-expressing cells, in comparison to control, showless axon retraction and more -βIII tubulin staining near the epicenter.Increased neuronal survival in the injured rats receiving Sp35 wasobserved.

The Sp35 retrovirus construct was made as follows: The Sp35 gene was PCRamplified using primers 5′-GATTACTCGAGATGCTGGCGGGGGGCGT GAGG-3′ (SEQ IDNO: 37), containing an XhoI site (underlined), and5′CGCGGGAATTCTCATATCATCTTCATGTTGAACTTG-3′ (SEQ ID NO: 38), containing anEcoRI site (underlined). The PCR product was digested with XhoI andEcoRI, then ligated into the Retrovirus vector pMIG (which containsIRES-GFP), which was previously cleaved with XhoI and EcoRI. The newvector was named pMMC078. All isolates of pMMC078 contained inadvertentpoint mutations, so two isolates of pMMC078 were ligated together.pMMC078.6 was cut with XhoI and AccI and pMMC078.7 was cut with XhoI andAccI. These two fragments were ligated together to make the finalcorrect plasmid, pMMC089. The DNA sequence of the insert was confirmedby DNA sequencing. Sp35 retrovirus was made as described. 293G cellswere split the day before transfection. 8 μg Sp35-retrovirus DNA wasused to transfect 5×10⁶ cells by lipofectamine (Invitrogen). Thecondition medium was harvested after 92 hours post-transfection. Theconditioned medium was centrifuged at 5000 g for 10 minutes, and thesupernatant used as a Sp35 retrovirus stock. This stock was stored at 4°C. for 1 week or −80° C. for 6 months.

Example 11 Animal Model of Spinal Cord Injury

All surgical procedures are performed using aseptic technique. For 1week prior to any surgical manipulation animals are handled. Ampicillin100 mg/kg SC is administered prophylactically prior to and after surgeryto reduce the incidence of bladder infection injury.

Animals are anesthetized using Midazolam at 2.5 mg/kg IP in conjunctionwith Isoflurane 2-3% in O₂ to deep anesthesia as measured by toe pinch.Animals are maintained on a circulating water heating pad for theduration of surgery and recovery. Ocular lubricant are used to preventcorneal drying and Atropine 0.05 mg/kg SC are given to reduce excesssalivation. A small incision is made in the skin and the muscleretracted to expose the vertebrae. A dorsal laminectomy at the spinallevel L6 (and L7 if placement of an intrathecal catheter is necessary,see below) is performed, L6/L7 and the adjacent spinous processesrigidly fixed in a spinal frame (David Kopf Instruments). A dorsalhemisection is performed at L6 with fine iridectomy scissors completelyinterrupting the main dorsomedial and the minor dorsolateralcoticospinal tract (CST) components. Following surgery, the laminectomysite is covered with a protective material such as Durafilm, and theoverlying muscle sutured with 4.0 chromic gut to protect the exposedspinal column. The skin is sutured and wiped with betadine solution.

Functional recovery of animals is evaluated using the Basso Beattie andBresnehan (BBB) scoring method commonly used to evaluate rats afterspinal cord injury. This method quantifies the hind limb function ofrats by detailed analysis of joint movement and weight bearing ability.Rats are evaluated the day after spinal cord injury then weeklythereafter.

Immediately after CST transection, adenovirus expressing Sp35 or GFP orcontrol virus (10¹⁰ particles) are injected at the site of transectionand regions immediately caudal and rostral to the injury site. A totalof 10 μl of Adv are injected at 5 different sites (4 ul/site). For theintrathecal administration of Sp35 protein, a small hole is made intothe dura of the spinal cord at 2 mm caudal to the lesion L7 and anintrathecal catheter is inserted into the subarachnoid space at L7. Thecatheter is slowly and gently slid above the spinal cord about 1 mmcaudal to the lesion. The portion of the catheter lying outside theintrathecal space is firmly sutured in place to the surrounding tissue.A primed mini-osmotic pump (Alza corp.) containing the test material(Sp35 protein or control protein) is connected to the exposed end of theguide cannula and inserted into the subcutaneous space. Followingsurgery, the laminectomy sites are covered with a protective materialsuch as Durafilm and the overlying muscle sutured with 4.0 chromic gutto protect the exposed spinal column. The skin is sutured and wiped withbetadine solution.

Histological Analysis: Tract tracing surgery occurs at the time of thesurgery to induce spinal cord injury. The skin on the head is shaved andwiped with Betadine and 70% alcohol. The animal is placed in astereotaxic frame. The scalp is incised longitudinally and theperiosteum is scraped from the calvaria. A hole is drilled in the skullapproximately 1-2 mm in diameter, and a glass microliter needle isinserted vertically into 8 locations in the motor cortex (coordinatesare determined according to the rat brain atlas of Paxinos and Waston,1997). Approximately 5 μl of tract tracer material (e.g., Biotin dextranamine, 10,000M. Wt) is injected and the needle is left in place for anadditional five minutes to allow diffusion of the solution. After needleremoval, the hole in the scull cap is plugged with gel foam and thescalp stapled closed over the injury site. Animals are allowed torecover and receive post-operative care (described below). Four to tenweeks later the animals are deeply anesthetized (Inactin 100-110 mg/kgip) and perfused for histology as described below. The tract tracer iscarried by anterograde transport mechanisms down the cortical spinaltract towards the caudal end of the spinal cord and provides a means toquantify anatomical connectivity within the corticospinal tract.

For immunohistochemistry experiments, animals are deeply anesthetizedwith Inactin (100-110 mg/kg IP) 2-8 weeks after surgery to induceinjury. The chest cavity is opened and the heart is exposed, to allowfor perfusion. A cannula is inserted into the left ventricle throughwhich 100 cc ice-cold PBS is pushed slowly (a hole will be cut in theright ventricle to allow fluid escape). This is followed by a slow butsteady drip of 4% paraformaldehyde (50-100 ml) until fixation ofeyes/ears/toes is obvious. Spinal cords are removed, with care tominimize alteration of the injury site, frozen in OCT, sectioned, andprocessed for immunohistochemistry. Other tissues optionally are alsocollected for later analysis. The animals receiving adenovirus Sp35showed increased axon sprouting as determined by βIII tubulin stainingfor neuron axon.

Example 12 Sp-35 Viral Vector Constructs

A pMIG-derived Sp-35 viral vector was made as follows. The full-lengthSp35 coding sequence was PCR amplified using primers 5′-GATTACTCGAGATGCTGGCGGGGGGCGTGAGG-3′ (SEQ ID NO: 37), containing an XhoI site, and5′CGCGGGAATTCTCATATCATCTTCATGTTGAACTTG-3′ (SEQ ID NO: 38), containing anEcoRI site. The PCR product was cut with XhoI and EcoRI, then ligatedinto the Retrovirus vector pMIG (Cheng et al, 1996, Nat. Biotechnol.145:576) which was cut with XhoI and EcoRI. This vector was designatedpMMC078. All isolates of pMMC078 contained point mutations, so twoisolates of pMMC078 were ligated together. Vector pMMC078.6 was cut withXhoI and AccI and pMMC078.7 was cut with XhoI and AccI. These twofragments were ligated to make the plasmid, pMMC089.

A pMIG-derived Sp35-HA viral vector was made as follows. A fragmentencoding Sp35 amino acids 326-614 in frame with the HA sequence wasobtained by using PCR with primers 5′-GCCTTCCGCGGCCTCAACTACCTGCGCGTGCTC-3′ (SEQ ID NO: 39), containing a SacII site, and 5′-CCGGAATTCTCAAGCGTAATCAGGAACGTCGTAAGGGTATATCATCTTCATGTTGAACTTGCG GGGCGCGTCGGC-3′ (SEQID NO: 40), with pMMC089 serving as a template. The longer primerincludes the HA coding sequence (italics) after Sp35 codon 614 andbefore the EcoRI site. The PCR product was then cut with Sac II andEcoRI, and used to replace the Sac II-EcoRI fragment containingwild-type Sp35 codons 326-614 in the pMIG-derived retroviral vector.

An Sp35-baculovirus HA vector was made as follows. The Sp35-HA codingsequence from Sp35-HA retroviral vector was cut out with XhoI and EcoRI,blunt-ended, and cloned into Bgl2-fill in site of baculo viral shuttlevector pBV-CZPG (U.S. Pat. Nos. 6,190,887; and 6,338,953), replacingLacZ gene under CMV promoter.

An Sp35-adenoviral vector was made as follows. Sp35-IRES-GFP codingsequence from the Sp35-retroviral was cut out with Xho I-fill in and NheI, then cloned into the EcoRI-fill in/Nhe I sites of the Adenovirusshuttle vector pDC315, under minimal CMV promoter.

Example 13 Animal Model of Remyelination

Female Long Evans rats are used in all studies. Rats are anaesthetizedusing Isoflurane and the T3/4 exposed and a dorsal hemi-laminectomyperformed. The chemical demyelinating agent, lysolecithin (3 μl of 1%lysolecithin in 0.9% saline), is then injected into the right side ofdorsal columns of the spinal cord 0.5-1 mm below the surface of thecord). Appropriate analgesic treatment is administered before and aftersurgery.

Three days later, the injection site is re-exposed (under isofluraneanesthesia, with appropriate analgesic treatment) and the followingtherapies injected into the injured spinal cord and an adenovirus vectorencoding protein Sp35/control protein is injected into the injury site.10¹⁰ particles of adenovirus encoding Sp35 or GFP control in a volume of10 μl will be injected into injured rat spinal cord in up to 5 differentsites in and around the site of lysolecithin-induced demyelination. Avolume of not greater than 2 μl is injected at each of the 5 injectionsites. For histological analysis of spinal corddemyelination/remyelination 2, 3, 4 or 6 weeks after surgery, animalsare deeply anesthetized with inactin (100-110 mg/kg ip) and perfusedwith fixative via the heart. The spinal cord is then removed andprocessed for analysis. The animal receiving Sp35 treatment showedincreased axon myelination as determined by IHC using anti-MBP proteinantibody or luxol fast blue.

Example 14 Sp35 RNAi

To address the role of Sp35 in brain function, we introduced thelentivirus Sp35 RNAi into postnatal 8 CGN cells. Sp35 RNAi infectedcells had shorter neurites and higher rates of proliferation thancontrol cells. These results indicate a role for Sp35 in regulating RhoAactivation.

Murine and rat Sp35 DNA sequences were compared to find homologousregions to use for candidate shRNAs. CH324 was constructed by annealingoligonucleotides LV1-035 and LV1-036 and ligating to Hpal and Xholdigested pLL3.7. The oligonucleotides were purchased from MWG. Thesequences are:

LV1-035 (sense oligo) (SEQ ID NO: 41)5′TGATCGTCATCCTGCTAGACTTCAAGAGAGTCTAGCAGGATGACGATC TTTTTTC LV1-036(antisense oligo) (SEQ ID NO: 3)5′TCGAGAAAAAAGATCGTCATCCTGCTAGACTCTCTTGAAGTCTAGCAG GATGACGATCA.

Prior to producing virus, DNA from pLL3.7 or candidate shRNA in pLL3.7were cotransfected with murine SP35-HA tagged plasmid at a ratio of 5 to1 into CHO cells in 6 well format. Knockdown was analysed by westernblot detection of SP35-HA tag from transfected CHO cell lysates as wellas by northern blot of total RNA prepared from duplicate wells. The blotwas probed with a 0.7 kb fragment of mSP35. Assays were performed 48hours post-transfection (data not shown). Viruses were produced from thebest candidate for use in rat neuronal cultures. The vector, additionalmethodology and virus production were as described in Rubinson et al. “Alentivirus-based system to functionally silence genes in primarymammalian cells, stem cells and transgenic mice by RNA interference.”Nat. Genet. 33, 401-6 (2003).

Example 15 RhoA Activation

COS7 cells co-expressing NgR1 and SP35 showed no changes in RhoA/GTPlevels in response to Omgp. This suggested that the SP35/NgR1 complex isnot sufficient for mediating signal transduction by a myelin inhibitor.

We explored the possibility that a ternary complex of SP35/NgR1/p75mediates signaling. Two approaches were used to evaluate interactionsbetween SP35, NgR1 and p75. First, binding was evaluated in a directbinding assay using an AP-SP35 conjugate. The AP-SP35 conjugate boundweakly to p75-expressing cells. AP-P75 bound to NgR1 expressing cells.The binding of AP-SP35 to NgR1 and p75 were measured by ELISA (FIG. 4).Second, binding of SP35 to NgR1 and p75 was evaluated by aco-immunoprecipitation from COS7 cells co-expressing SP35 NgR1 and p75.An anti-NgR1 antibody immunoprecipitated a complex containing SP35 andp75. An anti SP35 antibody also immunoprecipitated a complex containingp75. The interaction and co-immunopreicipitation data provided evidencefor a direct interaction between SP35, NgR1 and p75. We used confocalmicroscopy and antibodies against SP35, p75 and NgR1 to show that SP35,NgR1 and p75 co-localize to cell bodies and axons of p7 CG neurons fromrat.

Next we showed that the combination of SP35, NgR1 and p75 is sufficientfor the activities of the myelin inhibitor. Non-neuronal COS7 cells wereengineered to express all three components. Using these cells, we showedthat RhoA/GTP levels were up-regulated by Omgp. Omgp-Fc treatmentincreased RhoA/GTP levels in SP35/p75/NgR1 co-expressing cells, ascompared to other combinations of these three components. We confirmedexpression of the proteins by Western blotting of COS7 cells lysates.The affinity of myelin inhibitors binding to NgR1 were not affected bythe presence of p75 or p75 and SP35. The combined results support amodel whereby a ternary complex of NgR1, SP35 and p75 is required forRhoA regulation in the presence of NgR1 ligands (FIG. 5).

SP35 contains a cytoplasmic domain that has potential direct or indirectinvolvement in signaling. To determine the role of the cytoplasmicdomain, we produced a cytoplasmic domain truncation of SP35 (amino acids34 to 576 of SEQ ID NO: 2), to function in a dominant negative manner byforming an unproductive, ternary complex incapable of signaling. Wedesignated this molecule with the cytoplasmic domain truncation“DN-SP35” (for dominant negative SP35). We transfected postnatal day7(p7) CG neurons with full-length SP35 or DN-SP35, and then assayed forresponse to the inhibitory myelin components (Omgp; myelin and Nogo66).As shown in FIG. 6, DN-SP35 transfected cells failed to respond to theinhibitory myelin components and showed longer neurites than controls.In contrast, cells transfected with the full length SP35 constructshowed enhanced response to the inhibitory substrates, and had shorterneurites as compared to controls. This demonstrated that DN-SP35 acts asa competitor to attenuate neurite outgrowth inhibition caused by myelincomponents. We expected that exogenous, soluble SP35-Fc also would bindNgR1 and block the action of inhibitory substrates. As shown in FIG. 7,SP35-Fc reduced the neurite outgrowth inhibition by Omgp, Nogo66 andMAG.

Example 16 Neuroprotective Activity

Equal numbers of rat p6 cerebellar granule neurons was plated in eachwell of a 12-well cell culture plate in the presence or absence of 50 nMof sp35-Fc protein. These poly-D-lysine plates have been pre-coated[dried down] with 10 μg of CNS myelin, or 200 ng of Nogo66, MAG and Omgpor control-Fc. The neuronal cultures were maintained for 1-7 days at 37°C. and 5% CO₂. The neurons were healthy and grew well in the PBS controlwells independent of sp35-Fc treatment with full neurite extension[determined by neuronal specific marker, □III tubulin] as examined after3 days. In the absence of sp35-Fc, the neurons did not grow well in thewells coated with myelin, Nogo66, MAG and Omgp. There was minimumneurite sprouting [short and distorted] and the neurons did not appearto be healthy, with rounded cell body and condensed nuclear materials.DAPI staining demonstrated that the number of neurons detected in thesewells was less than that in the PBS control wells, suggesting neuronalloss. In the presence of sp35-Fc, long neurites were present and theneurons appeared healthy. DAPI staining demonstrated a higher neuronalnumber in these wells than those that did not receive the sp35-Fc. Thedata are summarized in Table 2 below.

TABLE 2 In the absence of sp35-Fc dried down substrate: OMgp/Nogo/MAG/Fc control myelin neurite extension short long distorted extended cellbody morphology rounded spread nuclear materials condensed clear neuronnumber at the end reduced same as control Fc of expt In the presence ofsp35-Fc dried down substrate: OMgp/Nogo/MAG/ Fc control myelin neuriteextension long long extended extended cell body morphology spread spreadnuclear materials clear clear neuron number at the end less reduced thatsame as control Fc of expt control FCThese data indicated that a soluble form of Sp35, e.g., Sp35-Fc,possesses neuroprotective activity.

In spinal cord hemi-transected (T9, SCT) rats, β-III tubulin staining ofthe spinal cord sections showed a substantial loss of neurons at thelesion site. A recombinant virus expressing sp35 was used to infect theSCT animals at the lesion site. Histological staining of these spinalcords showed an increased number of neurons around the lesion sitecompared to the control group that was infected with the vector virus.This is consistent with the in vitro experimental findings describedabove, and further indicates neuroprotective properties associated withSp35.

Example 17 Sp35 in Animal Model of Spinal Cord Injury

Since Sp35-Fc reduced neurite outgrowth inhibition caused by Omgp,Nogo-66 and MAG in vitro, we expected the molecule to promote functionalrecovery of CNS injuries in vivo. To confirm this, we administeredSp35-Fc to spinal chord hemisected rats, i.e., an animal model of acuteCNS trauma. As shown in FIG. 8 and FIG. 9, Sp35-Fc treated ratsdemonstrated significantly improved functional recovery, compared tocontrol rats treated with IgG.

Spinal cord injury and behavioral analysis were performed as follows.All surgical procedures were performed in accordance with the guidelinesof the Biogen Institutional Animal Use and Care Committee. Female LongEvans rats (190-210 g, Charles River, Wilmington, Mass.) wereanesthetized using 2.5 mg/kg Midazolam, I.P. and 2-3% Fluothane in O₂. Adorsal laminectomy was performed at spinal level T6 and T7. A dorsalhemisection was performed, completely interrupting the main dorsomedialand the minor dorsolateral corticospinal tract (CST) components.Immediately after CST transection an intrathecal catheter was insertedinto the subarachnoid space at T7 and connected to a primed mini-osmoticpump (Alzet model 2004) inserted into the subcutaneous space.Mini-osmotic pumps delivered Hu IgG isotype control protein (5 mg/ml,n=5, Pharmingen), PBS (n=3) soluble Hu Sp35-Ig fusion protein (4.3mg/ml, n=8) at a rate of 0.25 μl/h. Following surgery, the laminectomysite was sutured and the skin wound stapled closed. Postoperative careincluded analgesia (Buprenorphine 0.05 mg/kg s.c.) for 3 days andantibiotic treatment (Ampicillin 100 mg/kg s.c. twice daily) for 7 daysafter surgery. Bladders were expressed manually twice a day for theduration of the study (28 days) or until return of function (the time ofwhich was noted). All animals were blindly scored using the open-fieldBBB scoring system (Basso et al., 1995, J. Neurotrauma 12:1-21; Ono etal., 2003, J. Neurosci. 23:5887-5896). Rats were evaluated the day afterCST transection (day 2) and weekly thereafter for 4 weeks using theBasso-Beattie-Bresnahan (BBB) locomotor rating scale. Investigators wereblinded to the treatment groups for the duration of the study.

Example 18 Neuronal Survival and Axon Regeneration in the Rubro-SpinalTract (RST) Hemi-Section Injury Model

We also investigated the effects of Sp35 treatment on the regenerationof neurons in the rubro-spinal tract which directly contribute tolocomotion.

Adult 9-week-old Sprague-Dawley rats (200-250 g) were anesthetized withan intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8mg/kg). Under an operating microscope, a dorsal laminectomy wasperformed and the seventh thoracic spinal vertebra (C7) identified.After opening the dura mater, a right hemi-section was performed atspinal cord level C7 using a pair of spring scissors. Following spinalcord hemi-section, animals received a piece of gelfoam soaked witheither 10 μl of a 2 μg/ml solution of Sp35-Fc, or 10 μl of a 2 μg/mlsolution of human Ig, or 10 μl PBS, placed on top of the lesion site.After the operations, animals in each group were subdivided for axonaltracing and behavioral analysis. The animals for axonal tracing (n=5 foreach group) and behavioral analysis (n=7 for each group) were allowed tosurvive for 1 month.

Fluoro-Gold (FG, 6% w/v, Fluorochrome) was used to label the RST neuronsthat had regenerated their axons across the injury scar and reenteredthe caudal spinal cord. Two days prior to the end of the post-injurysurvival period (1 month), animals were anesthetized with anintraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg).A dorsal laminectomy was carried out and the T2 spinal segment wasidentified. FG at a volume of 0.5 ml was manually injected into theright T2 spinal cord using a Hamilton syringe. Two days later, theanimals were anesthetized and sacrificed with a lethal dose of ketamine(150 mg/kg) and xylazine (8 mg/kg) and they were perfusedintracardinally with normal saline, followed by 400 ml of fixativecontaining 4% paraformaldehyde in 0.1×PBS. The brains and spinal cordswere removed, postfixed with paraformaldehyde overnight, and then placedin 30% phosphate-buffered sucrose. Brain and spinal cord tissue were cutinto 30 mm sections on a cryostat and mounted onto gelatin-coatedslides. The number of FG-labeled RST neurons on the lesion side wasexpressed as a percentage of the total number of FG-labeled neurons onthe contra-lateral intact side. This percentage among groups wascompared statistically using one-way ANOVA followed by a Tukey-Kramermultiple comparisons test. As shown in Table 3, Sp35-Fc at 2 μg/mlpromoted the survival of rubro-spinal tract (RST) neurons.

TABLE 3 Percent Survival of RST Treatment Neurons (±S.E.M.) PBS 17.1 ±2   Sp35-Fc 31.9 ± 1.5 Control-Fc 14.5 ± 2.1

For behavioral analysis, the use of forelimbs during spontaneousvertical exploration was examined 1 month after different treatments asdescribed (Liu et al., 1999) with minor modifications. Rats were placedin a clear Plexiglas cylinder (15 cm in diameter and 30 cm high) thatencourages use of the forelimbs for vertical exploration for 5 min. Thefollowing behaviors were scored: (1) independent use of the left(unimpaired) or right (impaired) forelimbs for contacting the wall ofthe cylinder; and (2) simultaneous use of both forelimbs to contact thewall of the cylinder. The vertical exploration behavior was expressed interms of (1) percentage use of left (unimpaired) forelimb relative tothe total number of impaired, unimpaired, and both limb use; (2)percentage use of right (impaired) forelimb relative to the total numberof impaired, unimpaired, and both limb use; and (3) percentage use ofboth forelimbs relative to the total number of impaired, unimpaired, andboth limb use. The differences between groups were tested by one-wayANOVA followed by Bonferroni post hoc analysis. Sp35-Fc treated animalsshowed significantly improved front limb movement: 30% usage for bothforelimbs in Sp35-1-Fc treated animals versus 10% usage for bothforelimbs in control-Fc or PBS-treated animals; 55% left (unimpaired)limb usage versus 80% usage in control-Fc or PBS-treated animals; and29% right (impaired) limb usage versus approximately 15% in control-Fcor PBS treated animals.

Example 19 Sp35-Fc Promotes Retinal Ganglion Cell (RGC) Survival in theOptic Nerve Transection Model

We further confirmed the activity of Sp35 using the optic nervetransection model, which investigates factors that affect neuronalfunction. Young adult female Sprague Dawley (SD) rats were used in thisstudy. The right optic nerve of each animal was transectedintraorbitally 1.5 mm from the optic disc. A piece of gelfoam soakedwith 6% Fluoro-Gold (FG) was applied to the newly transected site rightbehind the optic disc to label the surviving retinal ganglion cells(RGCs). The animals were divided into 6 groups (n=6 in each group)receiving either Sp35-Fc, human IgG1, or just PBS, by intravitrealinjection. The volume of each intravitreal injection was 4 ml while thedosage of each injection was 2 mg. The intravitreal injections wereperformed immediately after the optic nerve transection.

All animals were allowed to survive for 1 week. Two days beforesacrificing the animals, the left optic nerve of each animal wastransected and 6% FG were used to label the surviving RGCs to serve asthe internal control. Animals were sacrificed with an overdose ofNembutal and the retinas dissected in 4% paraformaldehyde. Four radialcuts were made to divide the retinas into four quadrants (superior,inferior, nasal and temporal). The retinas were then post-fixed in thesame fixative for 1 hour before they were flat-mounted with the mountingmedium (Dako). The slides were examined under a fluorescence microscopeusing an ultra-violet filter (excitation wavelength=330-380 nm). LabeledRGCs were counted along the median line of each quadrants starting fromthe optic disc to the peripheral border of the retina at 500 mmintervals, under an eyepiece grid of 200×200 mm. The percentage ofsurviving RGCs resulting from each treatment was expressed by comparingthe number of surviving RGCs in the injured eyes with theircontra-lateral eyes. All data were expressed as mean±SEM. Statisticalsignificance was evaluated by one way ANOVA, followed by a Tukey-Kramerpost hoc test. Differences were considered significant for p<0.05.Sp35-Fc treated animals showed significant neuronal survival (83%) whencompared to control-Fc or PBS treated animals, which each only showedapproximately 50% neuronal survival.

OTHER EMBODIMENTS

Other embodiments are within the following claims.

1. An isolated polynucleotide encoding a soluble fragment of SEQ IDNO:2, wherein expression of said polynucleotide in a cell produces saidsoluble fragment of SEQ ID NO: 2 that is capable of decreasinginhibition of axonal growth of a central nervous system neuron.
 2. Theisolated polynucleotide of claim 1, wherein the a soluble fragment ofSEQ ID NO:2 is selected from the group consisting of: (a) a polypeptidecomprising amino acids 34-532 of SEQ ID NO:2; (b) a polypeptidecomprising amino acids 417-531 of SEQ ID NO:2; (c) a polypeptidecomprising amino acids 425-531 of SEQ ID NO:2; (d) a polypeptidecomprising amino acids 1-531 of SEQ ID NO:2; (e) a polypeptidecomprising amino acids 433-493 of SEQ ID NO:2; (f) a polypeptidecomprising an Sp35LRR domain, an Sp35 basic region C-terminal to the LRRdomain, and an Sp35 immunoglobulin (Ig) domain C-terminal to the basicregion, but lacks a transmembrane domain; (g) a polypeptide comprisingan Sp35 Ig domain, but lacking an Sp35 LRR domain, an Sp35 basic region,a transmembrane domain, and a cytoplasmic domain; (h) a polypeptidecomprising an Sp35 LRR domain, but lacking an Sp35 Ig domain, an Sp35basic region, a transmembrane domain, and a cytoplasmic domain, and (i)a polypeptide as in (f), further lacking a cytoplasmic domain; whereinsaid soluble fragment is capable of decreasing inhibition of axonalgrowth of a central nervous system neuron.
 3. The polynucleotide ofclaim 1, wherein said polynucleotide further encodes a heterologouspolypeptide fused to said soluble fragment of SEQ ID NO:2.
 4. Thepolynucleotide of claim 3, wherein said heterologous polypeptide isselected from the group consisting of an Ig polypeptide, a serum albuminpolypeptide, a targeting polypeptide, a reporter polypeptide, a humanNgR1 -binding polypeptide, one or more cysteine residues, and apurification-facilitating polypeptide.
 5. The polynucleotide of claim 4,wherein said heterologous polypeptide is selected from the groupconsisting of immunoglobulin Fc, human serum albumin or fragmentthereof; a histidine tag, an oligodendrocyte-myelin glycoprotein orfragment thereof, a myelin associated glycoprotein or fragment thereof,and a Nogo66 glycoprotein or fragment thereof.
 6. The isolatedpolynucleotide of claim 1, wherein the soluble fragment of SEQ ID NO:2is selected from the group consisting of: (a) a polypeptide consistingof amino acids 454-458 of SEQ ID NO:2; and (b) a polypeptide consistingof amino acids 453-458 of SEQ ID NO:2.
 7. The polynucleotide of claim 6,wherein said polynucleotide further encodes a heterologous polypeptidefused to said soluble fragment of SEQ ID NO:2.
 8. A compositioncomprising a pharmaceutically acceptable carrier and the polynucleotideof claim
 1. 9. A vector comprising the polynucleotide of claim
 1. 10.The vector of claim 9, wherein said polynucleotide is operatively linkedto an expression control sequence.
 11. The vector of claim 10, whereinsaid vector is a viral vector.
 12. The vector of claim 11, wherein saidviral vector is selected from the group consisting of an adenoviralvector, a lentiviral vector, a baculoviral vector, an Epstein Barr viralvector, a papovaviral vector, a vaccinia viral vector, and a herpessimplex viral vector.
 13. A host cell comprising the vector of claim 10.14. The host cell of claim 13, which expresses said soluble polypeptide.15. A method for producing an Sp35 polypeptide comprising culturing thehost cell of claim 14 and recovering said Sp35 polypeptide from theculture medium.
 16. An isolated polypeptide encoded by thepolynucleotide of claim
 1. 17. The polypeptide of claim 16, wherein saidpolypeptide is produced synthetically.
 18. The polypeptide of claim 16,wherein said polypeptide is conjugated to a polymer.
 19. The polypeptideof claim 18, wherein said polymer is selected from the group consistingof a polyalkylene glycol, a sugar polymer, and a polypeptide.
 20. Thepolypeptide of claim 19, wherein said polyalkylene glycol ispolyethylene glycol (PEG).
 21. The polypeptide of claim 18, wherein saidpolypeptide is conjugated to 1, 2, 3 or 4 polymers.
 22. The polypeptideof claim 21, wherein the total molecular weight of the polymers is from20,000 Da to 40,000 Da.
 23. An isolated polypeptide encoded by thepolynucleotide of claim
 3. 24. An isolated polypeptide encoded by thepolynucleotide of claim
 6. 25. The polypeptide of claim 24, wherein saidpolypeptide is cyclized.
 26. An isolated polypeptide encoded by thepolynucleotide of claim
 7. 27. A composition comprising apharmaceutically acceptable carrier and the polypeptide of claim
 16. 28.The composition of claim 27, further comprising a supplementary activecompound selected from the group consisting of an anti-NgR1 antibody orbinding fragment thereof and a soluble NgR1 polypeptide.
 29. Acomposition comprising a pharmaceutically acceptable carrier and thepolypeptide of claim 24.